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The Pharma Review

Section 2 - Analytical Method Development
Contents
Validated Method Development for Estimation of Atorvastatin Calcium and Fenofibrate in Fixed Dose Combination by HPTLC - P. B. Deshpande, G. Shridharan, Libi Anandi, D. Jadhav, M. C. Damle, S. V. Gandhi
THE UMBRELLA JOURNAL OF THE PHARMA INDUSTRY
3 6 9
Simultaneous High Performance Liquid Chromatographic Estimation of AmphotericinB in Dry Injection Dosage Form - J.P. Mall, P.C. Patel And Dr. J.N. Verma Stress Degradation Studies on Ceftazidime Sodium and Development of a Validated Stability-Indicating HPLC Assay - Lalitha N, S.B.Puranik, Sanjay Pai P.N, Rao G.K.
Spectrophotometric Methods for Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Tablet Dosage Form 12 - S. S. Kadukara, P. N. Ranjanea, S. S. Ranhera, S. V. Gandhia High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Amlodipine Besilate and Bisoprolol Fumarate in Pharmaceutical Preparations - R.B. Kakde, V.H. Kotak and D.L.Kale 14 Development And Validation of Spectrophotometric Method for Determination of Montelukast Sodium in Bulk and Tablet Formulation - Varun Pawar, Lalitha N, Puranik SB, Sanjay Pai P.N, Rao G.K. Gas Chromatographic Determination of Residual Solvents in Pharmaceutical Excipients - S. B. Puranik, Varun R. Pawar, N. Lalitha, P. N. Sanjay Pai, G. K. Rao UV-Spectrophotometry and First Order Derivative Methods for Estimation of Efavirenz in Bulk and Capsules - Charushila H. Bhirud, Atul A. Shirkhedkar, Ravindra A. Fursule and Sanjay J. Surana RP-HPLC Estimation of Eplerenone in Tablets - K. P. Bhusari, P. B. Khedekar, N. D. Amnerkar, S. M. Dhole, V. S. Banode Development and Validation of New RP-HPLC Method with UV-Detection for the Determination of Carvedilol in Human Serum - Mogallapalli L V Setti, Vijaya Ratna J Gas Chromatographic Determination of Methanol and Isopropyl Alcohol Impurities in Herbal Extracts - S. B. Puranik, Varun R. Pawar, Lalitha N, P. N. Sanjay Pai, G. K.Rao Simultaneous Spectrophotometric Estimation of Paracetamol and Tramadol Hydrochloride in Solid Dosage Form - P.G. Yeole, S.J. Wadher, M.P. Puranik, R.O. Ganjiwale First Order Derivative Spectrophotometric Determination of Nebivolol in Bulk and Tablets - Atul A. Shirkhedkar, Prasad M. Bugdane, Sanjay J. Surana 18 21 24 26 28 32 36 39
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Application of Oxidants to the Spectrophotometric Determination of Gemifloxacin Mesylate in Pharmaceutical Formulations - Marothu Vamsi Krishna, Dannana Gowri Sankar Simultaneous Estimation of Aceclofenac and Paracetamol in Combined Dosage Form by Two Wavelength Spectrophotometry - Wadher S. J., Momin M. Y., Puranik M. P. and Yeole P. G. Application of UV-Spectrophotometric Method for Estimation of Drotaverine Hydrochloride in Bulk & Tablets - A. A. Shirkhedkar, G. H. Upasani and S.J. Surana Oxidative Coupling, Complex Formation and Internal Salt Formation Reactions for Visible Spectrophotometric Determination of Alfuzosin Hydrochloride in Pharmaceutical Formulations - M. Vamsi Krishna, D. Gowri Sankar Development of Spectrophotometric Analysis Method of Atenolol Gel Formulations - Nandy B. C, Gupta R.N UV-Spectrophotometric Estimation of Nabumetone - M. N. Purohit, Sharad Mohan, Kunal Chokshi, G. V. Pujar Difference Spectroscopic Estimation of Raloxifene in Bulk and Formulations - Patel P. M., Patel R. C. and Patel N. M. Spectrophotometric Methods for the Determination of Rosuvastatin Calcium in Pure Form and in Pharmaceutical Formulations by Using Redox/Complexation Reactions - M. Vamsi Krishna and D. Gowri Sankar A Novel Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Frusemide in Tablets - R. K. Maheshwari Estimation of Fenoverine Hydrochloride in Pharmaceutical Dosage Forms by RP HPLC - Dr. K. Chitra, K. Sujatha, C. Vinodhini, B. Hareesh, B. Bharath, E. Brahma Reddy and B. Pamula Reddy Evaluation of Marketed Polyherbal Antidiabetic Formulations using Biomarker Charantin - P. M. Patel, N. M. Patel and R. K. Goyal Determination of Cefadroxil in Bulk Powder and its Dosage Forms by Spectrophotometric Method - Haresh M. Patel, Bhanubhai N. Suhagia, Shailesh A. Shah, and Ishwarsinh S. Rathod Reverse Phase HPLC Determination of Celecoxib in Dosage Forms - T. E. G. K. Murthy, Y. A. Chowdary, K. Narendra Kumar, D. Gowri Sankar and B. Durvasa Rao Simultaneous Estimation of Chlorzoxazone and Tramadol Hydrochloride in Tablet Formulation by Two Wavelength Spectrophotometry - M.P. Puranik, Anjali Hirudkar, S.J. Wadher, and P.G. Yeole Spectrophotometric Methods for Quantitative Estimation of Venlafaxine Hydrochloride from Tablet Formulation - S. Pillai and I. Singhvi New Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Ketoprofen in Tablet Dosage Form - R.K. Maheshwari Spectrophotometric Method for the Estimation of Valdecoxib and Paracetamol from Combined Dosage Form - K. E. V. Nagoji, V. Kiran Kumar, K. Ravi Sankar, V. Venkata Rao, Prafulla Kumar Sahu and M. E. B. Rao Simultaneous estimation of Pantoprazole and Domperidone in Pharmaceuticals Dosage Forms by RP-HPLC - Singh R. A., Arora Saurabh, Kumar Robin, Kumar Dinesh & Agarwal A.K H.P.L.C Method for Simultaneous Estimation of Tannic acid, Gallic acid, Chebulinic acid and Ethyl gallate in Terminalia chebula fruits - K. Jayaram Kumar, S.P. Bhatnagar Simultaneous Estimation of Cetirizine Hydrochloride and Salbutamol Sulphate in Pharmaceuticals dosage forms by RP-HPLC - Dr. R.A. Singh, Robin Kumar, Dinesh Kumar & A.K. Agrawal
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ANALYTICAL METHOD DEVELOPMENT
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Validated Method Development for Estimation of Atorvastatin Calcium and Fenofibrate in Fixed Dose Combination by HPTLC
P. B. Deshpande, G. Shridharan, Libi Anandi, D. Jadhav, M. C. Damle, S. V. Gandhi*
Abstract: A simple high-performance thin-layer chromatographic method has been developed and validated for determination of atorvastatin calcium and fenofibrate in a fixed dose combination. The drugs were separated on aluminum plates precoated with silica gel 60 F254 using chloroformmethanol (8:2 v/v) as solvent system. The drugs were well resolved with Rf values 0.29 and 0.77 for atorvastatin calcium and fenofibrate, respectively. Quantitative analysis was performed by densitometric scanning at 285 nm. The method was validated for linearity, accuracy, precision, specificity and robustness. The calibration plot was linear over the ranges 2001000 and 3201600 ng/band for atorvastatin calcium and fenofibrate, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.
Introduction
Atorvastatin calcium is a selective competitive inhibitor of the enzyme HMG-CoA reductase that catalyses conversion of HMGCoA to mevalonate, an important rate-limiting steps in cholesterol biosynthesis.1 Fenofibrate is a lipid lowering agent.2 Literature survey reveals that few HPLC and HPTLC methods have been reported for estimation of atorvastatin calcium3,4 and fenofibrate5,6 in combination with other drugs. One HPTLC method reported by Chaudhari et. al.7 for simultaneous estimation of atorvastatin calcium and fenofibrate used toluene:methanol: acetic acid (08:02:02 v/v/v) as solvent system. The limitation of this method primarily is high Rf value for fenofibrate (0.88) which makes precise densitometric evaluation rather difficult due to interference by solvent front.
fenofibrate were purchased from local market. All chemicals used were of analytical-grade. Preparation of Standard and Sample Solutions Atorvastatin calcium (10 mg) and fenofibrate (160 mg) were weighed separately, transferred to separate 10 ml volumetric flasks and dissolved in 10 ml of methanol. Stock solutions were further diluted with methanol to furnish working standard solution of concentration 100 ng/l for atorvastatin calcium and 160 ng/l for fenofibrate. For analysis of the tablet dosage form, the powdered sample equivalent to 10 mg atorvastatin calcium (160 mg fenofibrate) was accurately weighed and dissolved in 10 ml methanol. This solution was filtered and 1 ml of filtrate was further diluted to 10 ml with methanol to furnish solution of concentration 100 ng/l for atorvastatin calcium determination (Test Solution I). The 1 ml of second dilution was further diluted to 10 ml to furnish solution of concentration 160 ng/l of fenofibrate (Test Solution II). TLC Method and Chromatographic Conditions TLC was performed on 20 cm 20 cm aluminium-backed HPTLC plates coated with 0.2 mm layers of silica gel 60 F254 (E. Merck, Germany). Before use the plates were pre-washed with
Experimental
Chemicals and Reagents Pharmaceutical grade atorvastatin calcium and fenofibrate were obtained as gifts from Mepro Pharmaceuticals Pvt. Ltd., Wadhawan (Gujarat) and A TO Z Pharmaceuticals Pvt. Ltd., Chennai (Tamilnadu), respectively. Fixed dose combination tablets containing 10 mg of atorvastatin calcium and 160 mg of
Department of Pharmaceutical Analysis, AISSMS College of Pharmacy, Kennedy Road, Pune. *Author for correspondence e-mail: santoshvgandhi@rediffmail.com
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methanol, activated in an oven at 105OC for 20 min, then left to cool to room temperature. Solutions were applied to the plates, as 8 mm bands by means of a Camag Linomat IV semi-automatic sample applicator fitted with a 100-?l Hamilton syringe. Plates were then developed with 20 ml chloroformmethanol 8:2 (v/v) as mobile phase, in a 20 cm 20 cm Camag twin-trough chamber previously saturated for 20 min. The development distance was 15 cm (development time 20 min). After development the plates were removed from the chamber, dried in air and densitometric scanning at 285 nm in reflectance mode was performed with a Camag TLC Scanner-3 equipped with winCATs software version 1.4.2 incorporating track-position optimization. The slit dimensions were 6.00 mm 0.45 mm. Assay of the Marketed Formulation For assay of marketed formulation fixed volumes of test solution I and II (4 l for atorvastatin calcium and fenofibrate each) were applied to the plate which was then developed as described above. The amount of atorvastatin calcium and fenofibrate was determined from the standard calibration curve and the amount of each drug in sample was calculated. Validation of Method The method was validated in accordance with ICH guidelines for linearity, accuracy, specificity, intra-day and inter-day precision as well as robustness. For preparation of calibration plots aliquots of working standard solutions of atorvastatin calcium (100 ng/l) and fenofibrate (160 ng/l) were applied to the plates which were then chromatographed and processed as described under chromatographic conditions. To study the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50, 100, and 150 %. To study intra-day variation, six mixed standard solutions containing atorvastatin calcium (400 ng/band) and fenofibrate (640 ng/band) were prepared and applied to the plates. All solutions were analysed on the same day to record any intra-day variation in the results. To study inter-day variation, analysis of three mixed standard solutions of the same concentration was performed on three different days. To confirm the specificity of the method, solutions of atorvastatin calcium and fenofibrate and the extract from the tablet dosage form were applied to a TLC plate, plate was developed and scanned as described above. The effect of small, deliberate variation of the analytical conditions on the peak areas of the drugs was examined. Factors varied were volume of mobile phase ( 0.5 %), time from application to development (0, 10, 20, and 30 min) and
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from development to scanning (0, 30, 60, and 90 min). One factor at a time was changed to study the effect. The robustness of the method was checked at amount of 400 and 640 ng/band for atorvastatin calcium and fenofibrate, respectively.
Results and Discussion

As the HPTLC method reported by chaudhari et. al. for simultaneous HPTLC estimation of atorvastatin calcium and fenofibrate has very high Rf values particularly for fenofibrate (0.88), the objective of the current study was to develop a more accurate, precise, versatile, speedy and cost-effective HPTLC technique for determination of atorvastatin calcium and fenofibrate in fixed dose combination. Different mobile phases containing different proportions of chloroform, toluene, methanol, acetone, ethyl acetate, n-butanol and triethylamine were examined. The mixture of chloroformmethanol 8:2 (v/v) enabled satisfactory resolution of atorvastatin calcium and fenofibrate with good peak shape, Rf values of 0.29 0.019 and 0.77 0.025, respectively (Figure 1).
Standard calibration plots were linear over the range 200 1000 ng/band for atorvastatin calcium and 320 -1600 ng/band for fenofibrate with high correlation coefficient. Excipients present in the formulation did not interfere with the peaks of atorvastatin calcium and fenofibrate. The spectra of atorvastatin calcium and fenofibrate extracted from the tablets were also compared with the spectra obtained from atorvastatin calcium and fenofibrate standards and correlation was found to be good. The recovery was found to be 98.59-99.05 % for atorvastatin calcium and 99.15 99.32 % for fenofibrate. Intra-day variation, as RSD (%) was 0.640 for atorvastatin calcium and 0.412 for fenofibrate, with standard error 0.216 and 0.146, respectively. Interday variation, as RSD (%), was 0.710 for atorvastatin calcium and 0.580 for fenofibrate, with standard error 0.354 and 0.762, respectively. The method was also evaluated by assay of commercially
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available tablets containing atorvastatin calcium and fenofibrate. Six replicate analyses were performed on accurately weighed amounts of the tablets. The assay (%) was found to be 98.30 0.29 for atorvastatin calcium and 99.10 0.23 (Mean S.D.) for fenofibrate. Study of the robustness of the method revealed that peak areas were unaffected (RSD < 2 %) by small changes of the operating conditions.
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Bothara, Principal, AISSMS College of Pharmacy, Pune, India for providing infrastructure facilities to carry out this research work.
References
1. The Merck Index, 14th edition, Edited by Maryadele J. O'Neil, Merck Research Laboratories, Division of Merck and Con., Inc., Whitehouse Station, NJ, 2006, 864. Remingtons- The Science and Practice of Pharmacy, 21st Edition, Vol. II, Edited by Beringer P., Mack Publishing Co., Easton, PA, 2005, 1368. Erturk, S., Sevinc, E., Erosy, L. and Ficicioglu, S., J. Pharm. Biomed. Anal., 2003, 33, 1017. Rajeswari, K., Sankar, G. and Seshagirirao, J., Indian J. Pharm. Sci., 2006, 68, 275. Rani, S., Nivsarkar, M., Rathod, R., Guttikar, S. And Padh, A., Indian J. Pharm. Sci., 2005, 67, 297. EIGindy, A., Emara, S., Mesbah, M. and Hadad, G., Farmaco., 2005, 60, 425. Chaudhari, B. G. and Patel, N. M., Indian Drugs, 2007, 44, 378.
2. 3. 4. 5. 6. 7.
Conclusion
This validated HPTLC method can be used for routine analysis of atorvastatin calcium and fenofibrate in combined tablet dosage forms.
Acknowledgements
Authors are thankful to Mepro Pharmaceuticals Pvt. Ltd., Wadhwan (Gujarat) and A TO Z Pharmaceuticals Pvt. Ltd. (Chennai) for providing gift samples of atorvastatin calcium and fenofibrate, respectively and are also thankful to Dr. K. G.
Source: The Pharma Review, May 2009
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Simultaneous High Performance Liquid Chromatographic Estimation of AmphotericinB in Dry Injection Dosage Form
J.P. Mall*, P.C. Patel And Dr. J.N. Verma
Abstract: Simple, accurate and reproducible High Performance Liquid Chromatographic method, requiring no prior Instrumental methods have been developed for the estimation of Amphotericin B in Amphotericin B injection mentioned in I.P.2007,Volume2,Page No.727and728/USP 2007,Volume2,Page No.1410. In both monograph method of content estimation is determind by Microbiological assay of Antibiotics.As instrumental analysis is more accurate as well as less time consuming. Liquid Chromatographic method for the estimation of Amphotericin B was developed using Reversed phase H.P.L.C. having ultra violet detector and separation was carried out on Column C18(25X4.6mm,5Micron), using Water 486, mode by direct injection.. The retention time for standard and test was found to be 6.4 minutes.The method was validated according to ICH guidelines.The method described is simple, sensitive, reliable and reproducible for the quantitative estimation of active ingredient in dosage forms and their levels are found to be within the ICH limits.
Introduction
AmphotericinB is Antifungal agent, official in Indian Pharmacopoeia and United State Pharmacopoeia, chemically it is (3-amino-3,6-di deoxy-b-D-mannopyranosyloxy)-16-Carboxy3,5,8,9,11,13,15,35-octahydroxy-34,36-dimethyl-13,17-epoxyoctatviaconta-20,22,24,26,28,30,32-heptaen-37-olide and other antifungal polyenes produced by the growth of certain strains of Streptomyces nododus or by any other means. The review of literature revealed that no instrumental or chemical method is as yet reported for the simultaneous estimation of the drugs in dosage forms. This paper describes simple, rapid, accurate and reproducible method for determination of drugs in Injection dosage form.
Reagent: Acetonitrile and methanol were used of H.P.L.C. grade and Sodium dihydrogen ortho phosphate were used of A.R.grade.Other chemicals i.e. dimethylsulfoxide were used of A.R.grade.Mobile phase were prepared with distilled water and degassed in sonicator subject to filteration using Whatman filter paper No.41.In case of Standard and Sample preparation, it was filtered using 13 mm membrane filter of 0.2 micron pore size.
Procedure
Standard Preperation: Dissolve 50 mg. of Amphotericin B Working Standard in 50 ml. of dimethyl sulfoxide. Transfer 2.5 ml.from this prepared solution to another 50 ml. Volumetric flask, add 10 ml. dimethyl sulfoxide and make volume up to the mark with mobile phase, mix well. Test Preperation: As Amphotericin B for injection is a sterile freeze dried mixture of Amphotericin B and deoxycholate sodium with buffering agent and is filled in a sealed container. Dissolve content of vial using 20 ml.of distilled water. Transfer 1 ml.from this prepared solution to 50 ml. volumetric flask, add 10 ml.of dimethyl sulfoxide and make up volume to 50 ml. with mobile phase.
Experiment
Instrument: Water 486 Column C18 (25X4.6 mm, 5 Micron) Wavelength UV at 405 Flow Rate: 1.0 ml/ Minute
Lifecare Innovations. *Author for correspondence E-mail: sales@lifecareinnovations.com

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Blank: Transfer 10 ml. of dimethyl sulfoxide to 50 ml.volumetric flask and make up volume to 50 ml. with mobile phase. Final concentration of Standard and Sample preparation is about 50 mcg/ml. Claim: Each vial contains Amphotericin B- 50 mg. Mobile Phase: Mixture of Acetonitrile-Methanol and 0.01M Sodium dihydrogen orthophosphate in the ratio of 41: 10: 49 were used. Column: C18 (25X4.6 mm, 5 Micron) Wavelength: U V at 405 Flow Rate: 1.0ml/ Minute Both the dilutions of Standard and Sample preparation were injected in reversed phase H.P.L.C. having same condition and retention time for standard and test were observed same i.e. 6.4Minutes. Calculation:
Sample Area Standard Area X Standard dilution Sample dilution Potency of Std. 100
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Table 2 Results of Analysis of commercial injection by Microbiological Assay Method Sample AmphotericinB for Injection Label Claim mg/ml % of Label Claim Estimated 50.0 mg/ml 50.02mg/Vial(100.04%)
Results of analysis by both method were found almost same.
Recovery Studies
To study the accuracy, reproducibility and precision of the above proposed method , recovery studies were carried out by addition of Standard drug to pre analyzed sample. Results of recovery studies were found to be satisfactory.
S.N. Concentration of added Amount recovered % Recovery amount of drug 1. 2. 3.
0.53
5.0 mg. 10.0 mg. 15.0 mg.
4.90 mg. 9.94 mg. 15.02mg.
98.0 99.4 100.1
Au
0.24
4.75. 4.93
X Av.filled wt.
2.12
2.50, 2.71 2.88, 2.98 3.45, 3.73
Procedure for Analysis of Powder for Injection

Powder for Injection are sterile, solid substances, freeze-dried materials which are distributed in their final containers and which, when shaken with the prescribed volume of the appropriate sterile liquid, rapidly form clear and practically particle free solutions. In case of powder for injection 10 vials are selected at randomly and perform the content uniformity as per method described.
Table1 Sample AmphotericinB for Injection Label Claim mg/Vial % of Label Claim Estimated 50.0 mg/ml 50.15 mg/Vial(100.3%)
-0.06 0.00
7.68 8.03 8.26
5.59
9.83
Min
19.66
Typical Chromatogram of HPLC

The low value of Standard deviation and recovery was found close to 100%(limit98% to 101%), indicates the reproducibility and accuracy of the method. The method employing is very simple as well as rapid and accurate. and can be employed for routine analysis in this dosage forms using simple instrumentation. So this method can be employed for routine analysis in Quality Control laboratories.
Results of Analysis of commercial injection by proposed method

This is mean of five determination. In above case of Table1 standard deviation of peak area were observed about 1%, well within the standard limit of 2%. Simultaneous determination of drugs in Injection by Microbiological Assay method were observed. This is mean of five determination.
Acknowledgements
Authors are grateful to Managing Director-Lifecare Innovations for his co-operation
References
1. 2. Antimicrob Agents Chemother 1986 April;29(4):584-588.PMC 180446 J.ChromatogrB Biomed Sci Appl.2001 Sep 5,760(2):307-13.Related Articles Links.
Source: The Pharma Review, March 2009

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Stress Degradation Studies on Ceftazidime Sodium and Development of a Validated Stability-Indicating HPLC Assay
Lalitha N*, S.B.Puranik, Sanjay Pai P.N, Rao G.K.
Abstract: Ceftazidime was subjected to different ICH prescribed stress conditions. The drug was found more stable under neutral, photolytic, 3% oxidative and solid state conditions, while it was found to be less stable under acidic, alkaline and 30% oxidative conditions. A stability-indicating HPLC method was developed for analysis of the drug in the presence of the degradation products. It involved a C-18 column and a mobile phase composed of phosphate buffer pH 6.8 and methanol (10:3), which was pumped through the column in isocratic mode. The detection was carried out at 254 nm. The method was validated for linearity, range, precision, accuracy, specificity, selectivity and intermediate precision.
1. Introduction
The parent drug stability test guideline Q1A (R2) issued by International Conference on Harmonization (ICH)1 suggests that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to identification of degradation products and hence supporting the suitability of the proposed analytical procedures. It also requires that analytical test procedures for stability samples should be stability-indicating and they should be fully validated. Accordingly, the aims of the present study were to establish inherent stability of ceftazidime through stress studies under a variety of ICH recommended test conditions1 and,2 and to develop a stability-indicating assay.3 The drug is Pyridinium, 1-[[7-[[(2amino-4-thiazolyl)[(1-carboxy-1methyle-thoxy)imino]acetyl] amino]-2-carboxy-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-en-3yl]methyl]-,hydroxide,innersalt,pentahydrate,[6R-[6a,7b(Z)]] (Fig. 1).4,5 It is a white powder that is soluble in water and practically insoluble in alcohol. It is a semi synthetic 2nd generation cephalosporin with broad-spectrum, beta-lactamaseresistant available in reconstituted formulation. The literature survey reveals that methods are available for the mechanism of ceftazidime degradation in aqueous solutions,6 stability of ceftazidime pentahydrate in medicinal preparation biotium and ceftim,7 reversed-phase HPLC method for determination of the drug in pharmaceutical dosage forms,8
Fig. 1: Structural formula of Ceftazidime O S

H2N N NH N H H O O N
O
N
+
H 3C H3C
OH O
Determination of Ceftazidime by Thin-Layer Chromatography and Densitometry,9 First-derivative spectrophotometric and LC determination of ceftazidime and cefadroxil in urine,10 Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring comparison of HPLC with UV, single MSand tandem MS-detection.11 Accordingly, a stability-indicating method was developed, which could separate various degradation products.
2. Experimental
2.1. Materials Ceftazidime sodium was supplied by Karnataka Antibiotics Pharmaceuticals Ltd, Bangalore, India and used as such. Methanol (HPLC grade) was purchased from Qualigen Fine Chemicals, Mumbai, India. The other chemicals and solvents used in the studies were of analytical grade. Ultra-pure water, obtained from Millipak 0.22 m water purification systems.
Al-Ameen College of Pharmacy, Near Lalbagh Main Gate, Bangalore. *Author for correspondence E-mail: lallubalu@rediffmail.com
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2.2. Instrumentation Precision water baths equipped with MV controller (Julabo, Seelbach, Germany) were used for hydrolytic studies. Stability studies were carried out in humidity chamber (KBF 760, WTB Binder, Tuttlingen, Germany) and photo stability studies were carried out in photostability chamber designed Analytical Technologies Ltd. Bangalore using D65 and UV lamps. Thermal stability studies were performed in a dry air oven (NSW Limited, New Delhi, India). The mobile phase was degassed by Mark Ultrasonic sonicator. The HPLC system consisted of isocratic pumps (LC-10ALVP) and UV-visible dual-wavelength detector (SPD-10AVP), Winchrome-VP software (all from Shimadzu, Kyoto, Japan). The chromatographic separations were carried out on SS Accurasil (SGE) C-18 column of 250 mm 4.6 mm i.d. with particle size of 5 m. Additionally, intermediate precision studies were also carried out on Wakosil (SGE) C-18 column (250 mm 4.6 mm i.d., particle size 5 m). 2.3. Degradation studies All stress decomposition studies were performed at an initial drug concentration of 1 mg ml-1 in phosphate buffer (pH 6.8) and methanol (10:3). Acid hydrolysis was performed in 0.1M HCl at 80C for 30 min. The study in alkaline condition was carried out in 0.1 M NaOH at 80C for 10 min. For the study in neutral condition, drug dissolved in water was heated at 80C for 180 min. Oxidative studies were carried out at room temperature in 3% and 30% hydrogen peroxide for 24 h and 30 min respectively. Photodegradation studies were performed in water and in 0.1 M HCl. The solutions were exposed to D65 (60,000-70,000 lux) for 24 h. Suitable controls were kept under the dark. Additionally, the drug powder was exposed to dry heat at 50C for 8 d. Samples were withdrawn at appropriate time intervals and subjected to HPLC analysis after suitable dilution. 2.4. Separation studies HPLC studies were carried out first on all reaction solutions individually and then on a mixture of those solutions in which decomposition was observed. Separations were achieved by isocratic elution using phosphate buffer (pH 6.8) and methanol in the ratio of 10:3 as the mobile phase. The mobile phase was filtered through 0.22 m nylon membrane and degassed before use. The injection volume was 100 L and the mobile phase flow rate was kept constant at 0.5 ml min-1. The analyses were carried out at 254 nm. 2.5. Validation of the method 2.5.1. Linearity and range
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A stock solution of the drug was prepared at strength of 1 mg mL-1. It was diluted to prepare solutions containing 0.5 100 g mL-1 of the drug. The solutions were injected in triplicate into the HPLC column, keeping the injection volume constant (100 L) 2.5.2. Precision Six injections, of three different concentrations (5, 10 and 20 g mL-1), were given on the same day and the values of relative standard deviation (R.S.D.) were calculated to determine intraday precision. These studies were also repeated on different days to determine inter-day precision. 2.5.3. Accuracy Accuracy was evaluated by fortifying a mixture of degraded solutions with four known concentrations of the drug. The recovery of the added drug was determined. 2.5.4. Specificity and selectivity The specificity of the method was established through study of resolution factors of the drug peak from the nearest resolving peak, and also among all other peaks. 2.5.5. Intermediate precision The intermediate precision was established through a study on a different chromatographic system using a different column.
3. Results and discussion

3.1. Degradation behavior HPLC studies on Ceftazidime under different stress conditions suggested the following degradation behavior: 3.1.1. Acidic condition The drug gradually decreased with time on heating at 80C in 0.1 M HCl, forming degradation products at RT 1.83 and 3.1 in 20 min. At the end of 30 min the drug was completely degraded. 3.1.2. Neutral (water) condition Upon heating the drug solution in water at 80C for 30 min, almost 20% of drug was degraded. At the end of 180 min, complete degradation of the drug was observed with the corresponding rise in the major degradation peaks at RT 1.81, 2.64 and 3.04 min. 3.1.3. Degradation in alkali The reaction in 0.1 M NaOH at 80C was so fast that whole of the drug was degraded in 10 min. Drug degradation was associated with rise in a major degradation product at RT 1.92, 2.6 and 3 min.
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3.1.4. Oxidative conditions In 3% H2O2, the drug was degraded within 12 h and completely degraded at the end of 24 h. Drug was degraded completely at the end of 30 min in 30% H2O2. The oxidative degradation of drug was associated with rise in major degradation products at RT 1.9 and 3.14 min. 3.1.5. Photolytic conditions Major degradation products were observed after exposure of drug solution in 0.1 M HCl to D65 for 12 h at RT 1.8 and 3.1 were formed. The nature of degradation in light and dark was found to be similar, indicating that light had no effect on the degradation of the drug in acid. On the other hand, the samples in water degraded under D65 for 24 h to a major product at RT 2.7, along with minor degradation products at RT 1.87 and 3.14. Corresponding rate of degradation in dark was much slower. 3.1.6. Solid-state study The solid-state studies showed that ceftazidime was stable to the effect of temperature. When the drug powder was exposed to dry heat at 50 C for 8 d, 20.85% drug was degraded without giving any degradation product. 3.2. Development and optimization of the stability-indicating method The method was optimized to separate major degradation products formed under various conditions. Resolution was also checked on mixture of the degradation solutions to confirm the separation behavior. The resulting chromatogram is shown in Fig. 2. It indicates that the isocratic method was successful in separation of drug and all chromophoric degradation products. 3.3. Validation of developed stability-indicating method The response for the drug was strictly linear in the concentration range between 5 and 100 g mL-1. The mean ( %R.S.D.) values of slope, intercept and correlation coefficient were 22662 (1.13), 28804 (1.805) and 0.9977 (0.015), respectively. The data obtained from precision experiments are given in Table 1 for intra-and inter-day precision studies. The %R.S.D. values for intra-day precision study were <1.0% and for inter-day study were <2.0%, confirming that the method was sufficiently precise. Percentage recovery was calculated from differences between the peak areas obtained for fortified and unfortified solutions. As shown from the data in Table 2, excellent recoveries were made at each added concentration.
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Fig. 2: Chromatogram of mixture of degradation solutions of Ceftazidime

Method File Detector Date Run Type of Analysis
Pk.Wdth 4
: : : : :
C:\LALITHA.MET UV-VIS. 20 Dec. 2007 ch1: 21 Percent On Area

Area Rej. 5
System Time
: HPLC :
Peak Thrsh. 30
HL.Rej. 4
Peaks at 1.81 and 3.4 are observed in all stress degradation conditions. Peak at 2.64 observed in neutral and alkaline conditions. The standard drug ceftazidime eluted at 6.78 min. Table 1: Reproducibility and precision data evaluated through intra-day and inter-day studies Actual Intra-day measured Inter-day measured concentration concentration (g ml-1) concentration (g ml-1) (g ml-1) S.D.; R.S.D.% (n = 6) S.D.; R.S.D.% (n = 3) 5 10 20 5.132 0.0511; 0.997 9.843 0.0758; 0.769 19.975 0.0952; 0.477 5.738 0.0189; 0.331 9.745 0.0828; 0.850 19.326 0.198; 1.025
Table 2: Recovery studies Actual concentration (g mL-1) 5 10 20 50 Calculated concentration (g mL-1) S.D.; R.S.D.% (n = 3) 5.103 0.0372; 0.738 10.351 0.0477; 0.459 20.444 0.1676; 0.820 49.968 0.768; 1.537 Recovery (%)
102.06 103.51 102.22 99.93
Fig. 2 shows that the method was sufficiently specific to the drug. The resolution factor for the drug peak was >3 from the nearest resolving peak. Intermediate precision was performed to confirm that separation was satisfactory under conditions external to the method. Good separations were always achieved, indicating that the method remained selective for all components under the tested conditions.
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ANALYTICAL METHOD DEVELOPMENT 4. Conclusions

The study shows that ceftazidime is sensitive to acidic, alkaline and 30% oxidative conditions. It is more stable to 3% oxidative, neutral, photolytic and solid-state condition. A stability-indicating method was developed, which separates all the degradation products formed under variety of conditions. The method proved to be simple, accurate, precise, specific and selective. Hence it is recommended for analysis of the drug and degradation products in stability samples by the industry.
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References
1. 2. 3. 4. 5. 6. 7. 8. 9. ICH, Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, IFPMA, Geneva, 2003. S. Singh 1, M. Bakshi 2, Pharm. Tech. On-line 24 (2000), 1. M. Bakshi 1, S. Singh 2, J. Pharm. Biomed. Anal. 28 (2002), 1011. http://www.rxlist.com/cgi/generic/Ceftazidime.htm, 2005 (accessed on 15.12.2005). USP-27-NF, The United States Pharmacopoeial Convection Inc. Rockville, MD, 2004, p.357. M. Zajac 1, J. Siwek 2, I. Muszalska 3, Acta Pol Pharm 55(4) (1998), 275. M. Zajac 1, A. Jelinska 2, A. Sobczak 3, W. Musial 4, Acta Pol Pharm, 62(1) (2005), 11. S Joshi 1, C.J. Sciacchitano 2, B. Mopper 3, J.J. Specchio 4, J Biomed Sci and Appl , 657(2) (1994), 395. J. Krzek 1, M. Dabrowska-Tylka 2, Chromatographia, 58 (2003), 231.
10. A. El-Gindy 1, M. A. Fattah 2. El Walily 3, M. F. Bedair 4, J Pharm Biomed Anal. 23(2) (2000) 341. 11. J. Tuerk 1, M. Reinders 2, D. Dreyer 3, T. K. Kiffmeyer 4, K. G. Schmidt 5, H. M. Kuss 6, J.Chorma.B, 831(1-2) (2006) 72.
Source: The Pharma Review, March 2009
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Spectrophotometric Methods for Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Tablet Dosage Form
S. S. Kadukara, P. N. Ranjanea, S. S. Ranhera, S. V. Gandhia*
Abstract: This work is concerned with the determination of Olmesartan medoxomil and Hydrochlorothiazide in marketed formulation by simultaneous equation method, absorbance correction method and area under the curve method. The linearity was found in the range 8-40 g/mL and 4-20 g/mL for Olmesartan medoxomil and Hydrochlorothiazide respectively for all the three methods. The developed three spectrophotometric methods were validated statistically and can be used for routine simultaneous determination of these drugs in quality control laboratories.
Introduction
Olmesartan medoxomil (OM, 2, 3-dihydroxy-2-butenyl 4-(1hydroxy-1-Methyl ethyl)-2-propyl-1- [p- (o-1H-tetrazol-5ylphenyl) benzyl] imidazole-5-carboxylate, cyclic 2, 3-carbonate) is a selective AT1 subtype angiotensin II receptor antagonist.1 Hydrochlorothiazide (HTZ, 6-chloro-3,4-dihydro-2H-1,2,4benzothiadiazine-7-sulphonamide-1,1-dioxide) is a diuretic antihypertensive agent.2 Extensive literature survey revealed UV-Vis spectrophotometric determination of OM in pharmaceutical dosage form.3 HPLC-MS4 and LC-MS5 determination of OM has also been reported in biological samples. Several assay methods have been reported for determination of HTZ in pharmaceutical dosage form or in biological samples as a single or in combination with other drugs. These include spectrophotometry, 6 RP-HPLC, 7 HPLCNarrowbore chromatography,8-9 HPTLC10 and electrochemical study.11 Also stability-indicating LC method has been reported for HTZ determination in combination with ramipril.12 No reports were found for simultaneous spectrophotometric determination of OM and HTZ in combined tablet dosage form. Aim of the present work was to develop simple, economical, rapid, accurate and precise spectrophotometric methods for determination of OM in combination with HTZ in pharmaceutical formulation. The proposed methods were optimized and validated as per the International Conference on Harmonization (ICH) guidelines.13
Materials and Methods

Instrumentation The instrument used in the present study was JASCO double beam UV/visible spectrophotometer (Model UV-550) connected to a computer with spectra manager software (Version 1.54A). The slit width was 2 nm and wavelength scanning speed being 400 nm/min. The absorption spectra of the test and reference solutions were recorded in 1 cm matched quartz cells over the range 200-400 nm. All weighing were done on electronic balance (Model Shimadzu AY -120). Reagents and Chemicals Methanol (UV grade) was purchased from Merck specialties Pvt. Ltd. (Mumbai, India). OM and HTZ was kindly supplied by Micro Labs Ltd. (Hosur, TN, India) and used as such without further purification. OLMAT-H tablets (Micro Labs Ltd.) labeled to contain OM 20 mg and HTZ 12.5 mg was purchased from local market. Preparation of Standard Stock Solutions Pure drug samples of OM and HTZ were dissolved separately in methanol to give stock solution of 0.2 mg/mL and 0.1 mg/mL, respectively. For each drug different aliquots were pipetted out from standard stock solution into series of 10 mL volumetric flask and diluted up to the mark using distilled water to determine linearity and range. Preparation of Sample Stock Solution Twenty tablets were weighed accurately and powdered. Powder
Department of Pharmaceutical Analysis, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Pune. *Author for correspondence E-mail: santoshvgandhi@rediffmail.com
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equivalent to 10 mg of HTZ was weighed and transferred to 100 mL volumetric flask and dissolved in methanol by shaking the flask for 10 minutes. The solution was filtered through Whatman filter paper no. 41 and 0.5 mL of filtrate was further diluted to 10 mL with distilled water to obtain final concentration 5 g/mL of HTZ (8 g/mL of OM). Recovery studies The accuracy of the proposed method was checked by recovery studies, by addition of standard drug solution to preanalysed sample solution at three different concentration levels within the range of linearity for both the drugs.
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wavelength. The equations obtained for the determination of concentration are CHTZ = A 317.4/9.671 ---------------(3)
COM = A 256 - (21.312 * CHTZ)/ 35.312 ---------------(4) Area Under the Curve Method (Method III) This method involves area calculation of integrated value of absorbance in the indicated region of wavelengths. Spectrums of both the drugs were recorded and area under the curve was calculated employing data processing mode. The optimum wavelength regions 254-258 nm and 271-273 nm were selected for area calculation on the basis of repeated observations, which showed good linearity of AUC against concentration range. The X values were calculated at these wavelength regions. Where, X = AUC of component between selected wavelengths/ Concentration in gm/L The X values of OM were 132.02 and 42.81 while of HTZ were 64.01 and 115.72 at 254-258 nm and at 271-273 nm, respectively. The concentration of each drug was then calculated by using following equations. COM= 115.72 * AUC 254-258 - 64.01 * AUC 271-273/- 12537.09 ---- (5) CHTZ = 42.81 * AUC254-258 - 132.02 * AUC 271-273/- 12537.09 ---- (6) where AUC254-258 and AUC271-272.6 are the area under the curve of sample in the selected wavelength region.
Theory
Simultaneous Equation Method (Method I) The Figure 1 shows zero order overlain absorption spectra of OM and HTZ. Wavelengths 256 nm (lmax of OM) and 272 nm (lmax of HTZ) were selected for derivation of equations. The absorptivities of OM and HTZ were 32.27 (ax1) and 18.22 (ay1) at 256 nm (l1) and 21.44 (ax2) and 60.61 (ay2) at 272 nm (l2), respectively. A1 and A2 are absorbance of diluted sample at 256 nm and 272 nm respectively. The concentration of each drug was then calculated by using equations (1) and (2) given below. COM = (A2ay1-A1ay2) / (ax2ay1-ax1ay2) CHTZ = (A1ax2-A2ax1) / (ax2ay1-ax1ay2)
1.8 1.5
-------- (1) -------- (2)

The linearity was found to be in the range 8-40 g/mL and 4-20 g/mL for OM and HTZ respectively with high correlation coefficient (> 0.998) for all the three methods. The proposed methods were also evaluated by the assay of commercially available tablets containing OM and HTZ (n = 6). The % assay represented as mean S.D. (% RSD) of commercial formulation by three methods was found to be 101.62 0.401. (0.394), 100.14 0.507. (0.506), 100.49 0.018. (0.017) and 100.85 0.854. (0.847), 101.61 0.054. (0.053), 100.23 0.489 (0.488) for OM and HTZ by method I, II and III respectively For recovery study different volumes of standard stock solution were added to the fixed volume of sample solution at 50 %, 100 % and 150 % level. The total amount of drug added was determined by proposed methods, results of which are shown in Table 1.
Abs 1
0.5 0 200
OM HTZ
250
300 Wave length (nm)
350
400
Fig. 1: Zero order overlain spectra of OM (16g/mL) and HTZ (16 g/mL)
Absorbance Correction Method (Method II) This method involves absorbance correction for OM determination by subtracting absorbance of HTZ from total absorbance of sample at 256 nm (max of OM). Since at 317.4 nm OM shows nearly zero absorbance; HTZ concentration is directly determined from absorbance of sample at this
Conclusions
The validated spectrophotometric methods can be used for routine determination of OM and HTZ in combined tablet dosage form.
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Table 1: Recovery studies of OM and HTZ Drug Conc. of drug added Method I
a
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2.
Method II % RSD % Recovery S.D.

a
Method III % % Recovery RSD S.D.

a
http://en.wikipedia.org/wiki/Hydrochlorothiazide. accessed on 28.07.2007 Celebier, M. and Altinoz, S., Pharmazie, 2007, 62, 419. Yu, Y., Hou, Y.N., Liu, J.F. and Wang.J., J. Chin. Pharm, 2006, 41, 1592. Liu, D., Hu, P., Matsushima, N., Li, X., Li, L. and Jiang, J. J. Chromatogr. B., 2007,856, 190. Lande, N. R., Shetkar,B. M., Kadam, S. S. and Daneshwar, S. R., Indian drug, 2000, 37, 574. Suhagia, B.N., Shah, R.R. and Patel, D.M., Indian J. Pharm. Sci., 2005, 67, 37. Farthing, D., Fakhry, I., Elizabeht, B.D., Ripley, D.S., J. Pharm. Biomed. Anal., 1998, 17, 1455.
3.
g/ % % Recovery ml Level S.D. OM 4 8 12 HTZ 2.5 5 12.5

a
% RSD
4. 5. 6. 7. 8.
50
99.640.826
0.829 100.290.302 0.301 100.660.161 0.160 0.632 100.460.532 0.530 100.350.893 0.890
100 99.580.629 50
150 100.550.679 0.676 100.310.211 0.211 100.690.980 0.974 100.610.826 0.821 101.610.982 0.975 100.230.718 0.716 0.612 100.540.597 0.594 99.960.227 0.227 0.953 100 99.280.607
150 100.150.404 0.404 100.630.195 0.194 99.690.950
average of three determinations
Acknowledgment
The authors are thankful to Micro Labs Ltd. for providing the pure drug samples. Thanks are also extended to Principal, Dr. K. G. Bothara, AISSMS College of Pharmacy for providing infrastructure facilities.
9. Zendelovska, D., Stafilov, T. and Milosevski, P., Biomed. Chromatogram., 2004, 18, 71. 10. Shah, N.J., Suhagia, B.N., Shah, R.R.and Patel, D.M., Indian J. Pharm. Sci., 2007, 69, 240. 11. Razak, O.A. J. Pharm. Biomed. Anal., 2004, 34,433. 12. Belal, F., Al-Zaagi, I.A., Gadkariem, E.A. and Abounassif, M.A. J. Pharm. Biomed. Anal., 2001, 24 , 335. 13. ICH Harmonised Tripartite Guideline (Nov. 2005) Validation of Analytical Procedures: Text and Methodology Q2 (R1).
References
1. http://www.pharmmarketing.com/bulk_drug_eng.htm. accessed on 30.07.2007
Source: The Pharma Review, December 2008
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High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Amlodipine Besilate and Bisoprolol Fumarate in Pharmaceutical Preparations
R.B. Kakde*, V.H. Kotak and D.L.Kale
Abstract: A novel analytical method for simultaneous determination of amlodipine besilate and bisoprolol fumarate in their combined dosage form has been developed by HPTLC procedure, using ethyl acetate : methanol : ammonia (6:0.5:0.5 v/v/v) as mobile phase and Merck HPTLC plate precoated 60 F254 silica gel on aluminum sheet as stationary phase. Detection was carried out densitrometrically using UV detector at 229 nm. The retention factor (RF) of amlodipine besilate and bisoprolol fumarate was 0.390.02 and 0.520.02 respectively. The linear regression analysis data was used for the regression line in the range of 500-1000 ng/spot for both amlodipine besilate and bisoprolol fumarate with correlation coefficient value close to 1.0. The developed method was validated using various validation parameters such as accuracy, precision, specificity, limit of detection, limit of quantitation and ruggedness, which make method as choice for routine quality control analysis for simultaneous estimation of amlodipine besilate and bisoprolol fumarate in their combined formulation.
Introduction
Amlodipine (a dihydropyridine) is a calcium-channel blocker. It is given as besilate and used in the management of hypertension and angina pectoris. It is chemically 3-ethyl-5-methyl-2-(2aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6methylpyridine-3,5-dicarboxylate monobenzenesulphonate. It is official in BP.1 Bisoprolol is a cardioselective beta blocker. It is given as the fumarate in the management of hypertension and angina pectoris. Chemically it is ()-1-[4-[[2-(1-methylethoxy) ethoxy] methyl] phenoxyl-3-[(1-methyl ethyl) amino]-2-propanol.It is official in USP.2 Literature survey reveals the availability of several UV,3-6 HPLC7-10 and HPTLC10 methods for estimation of amlodipine besilate (AMLO) as alone or in combination with other drugs and bisoprolol fumarate (BISO) can be determined by UV,11 RPHPLC,12 HPTLC13 in combination with hydrochlorthiazide and RPHPLC14 as alone is reported. Present work emphasizes on the quantitative estimation of amlodipine besilate and bisoprolol fumarate in their combined dosage form by HPTLC.
Experimental
Preparation of Standard Solutions Standard stock solution was prepared by dissolving 69.34 mg of amlodipine besilate (equivalent to 50 mg of amlodipine) and 50 mg bisoprolol fumarate in 50 mL of methanol. Standard solution was prepared by diluting 5 mL of this solution to 50-mL with methanol to get final concentration of 100 g mL-1 for both the drugs. Preparation of Sample Solution Twenty tablets were accurately weighed and average weight was calculated. The tablets were then crushed to obtain fine powder; an accurately weighed quantity of tablet powder equivalent to about 5 mg amlodipine was transferred to 50 mL volumetric flask and shaken with 15-20 mL methanol for 30 minutes for dissolving active ingredients completely, then volume was made up to the mark with methanol, the solution was mixed and filtered through Whatman Grade I filter paper. The concentration of final solution was 100 g mL-1 for both drugs.
University Department of Pharmaceutical Sciences, RTM Nagpur University, Nagpur. *Author for correspondence E-mail: drkakde@yahoo.com
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Thin-Layer Chromatographic Analysis The standard solution was applied in the form of bands of 4 mm width with space of 4 mm on the precoated silica gel aluminium plate 60 F254 (10cm10cm) in the range of 1-10 L with the help of microliter syringe using LINOMAT-IV automatic sample applicator. The mobile phase consists of methanol: ethyl acetate: ammonia (0.5:6:0.5, v/v/v). Linear ascending development was carried out in 10 cm 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland). The optimized chamber saturation time for mobile phase was 20 minutes at room temperature (25 2OC) at relative humidity of 60 5%. The length of chromatogram run was 70 mm. Subsequent to the development; TLC plates were dried in a current of dry air with the help of an air dryer. Densitometric scanning was performed on Camag TLC scanner III in reflectance-absorbance mode at 229 nm (Fig.1) for all measurements and operated by WINCATS 4.0 software.
100.0 [AU] 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 100.0 [AU] 80.0 70.0
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For specificity studies sample solutions were prepared by exposing an accurately weighed quantity of tablet powder equivalent to about 5 mg of amlodipine to various stress conditions for 24 hrs like at 50OC after addition of 1.0 mL of 0.1 N HCl (acid), at 50OC after addition of 1.0 mL of 0.1 N NaOH (alkali), at 50OC after addition of 1.0 mL of 3.0% H2O2 (oxide), at 60OC and in UV-cabinet at 265 nm.
Result and Discussion

Analytical Parameters and Validation The method was validated for accuracy, precision, specificity and ruggedness. Linearity was determined at different concentrations, amlodipine besilate showed good correlation coefficient in the concentration range of 500-1000 ng/spot (r = 0.999 and 0.999 by height and area respectively) and bisoprolol fumarate in the range of 5001000 ng/spot (r = 0.997 and 0.998 by height and area respectively).The correlation coefficient value is close to 1.0 shows that amlodipine besilate and bisoprolol fumarate follows linearity with varying concentration. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve as per ICH15 guidelines. System reproducibility was determined by five replicate applications and five times measurement of a laboratory mixture at the analytical concentration. The reproducibility of sample with respect to height and area for active compounds were expressed in terms of S.D. and %R.S.D. The spots at RF 0.39 (Amlodipine besilate) and 0.52 (Bisoprolol fumarate) were observed in the densitograms of the drug samples of tablets (Fig.2). There was no interference from the common excipients present in tablets.
600 500
Detector Response
BISO AMLO
60.0 50.0 40.0 30.0 20.0 10.0 0.0
200.0 229 250.0
300.0 Wave length (nm)
[nm]
400.0
Fig. 1: Overlain UV Spectra of Amlodipine Besilate and Bisoprold Fumarate for selection of suitable wave length (229nm) by scanning.
The source of radiation utilized was deuterium lamp. Evaluation was done via peak height and peak area. The linear regression analysis data was used for the regression line in the range of 500-1000 ng/spot for both the drugs. Precision of the method was determined by analyzing the marketed formulations. Accuracy of the proposed method was ascertained by carrying out recovery studies by standard addition method. To fixed amount of market formulation different concentrations of mixed pure drug solution of amlodipine besilate and bisoprolol fumarate were added. The sample solutions containing market formulation and pure drug solution were in the range of 80-120% of the concentration mentioned under sample preparation for tablets.
Detector Response
400 300 200 100 0 0.01 0.21 0.41 0.61 0.81 1.101
Rf valve A - Amlodipine besilate B - Bisoprolol fumarate Fig. 2: Typical densitograms of AB and BF

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The results of recovery studies as evaluated by height and area were found to be close to 100% which signifies the accuracy of the method. Specificity studies results reveals that both the drugs degraded in presence of peroxide and this was confirmed by decrease in height and area of both the drugs, when compared with the spot of standard solution but there was no change in RF value. Ruggedness was performed under three different conditions different days, different analysts and intra day. The results show the %RSD values <2.0% signifies the precision of the method.
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References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. British Pharmacopoeia Vol. 1, Department of Health Social Services and Public Safety, H.M. Stationary Office London, 2004, p. 126 United States Pharmacopoeia, 24th Ed., United State Pharmacopoeial Convention, Inc., Rockville, MD., 2000, p. 268. K. Gohil, P. Trivedi, and K. Molvi , Indian J. Pharm. Sci., 67, 376 (2005) P.R. Topale, N.J. Gaikwad, and M.R. Tajne, Indian Drugs, 40, 119121(2003) Nafisur Rahman, and Md. Nasrul Hoda, J.Pharm. Biomed. Anal., 31, 381-392 (2003) Nafisur Rahman, and Syed Najmul Hejaz Azmi, Farmaco, 56, 731-735 (2001) J.R. Rao, S.S. Kadam, and K.R.Mahadik, Indian Drugs, 39, 378 (2002) A.Zarghi, S.M. Foroutan, A.Shafaati and A.Khoddon, Farmaco, 60, 789-792 (2005) A.P.Kulkarni, G.V. Gat, S.V. Pimple, and M.A.Joshi, Indian Drugs, 40, 298 (2003) Martin Josefsson, Anna-Lena Zackrisson and Bjrn Norlander, J. Chromatogr B: Biomed. Sci. Appl., 672, 310-313 (1995) R. Sahu, and V.B. Patel, Indian J. Pharma. Educ. and Research, 68, 764-767 (2006) L.J.Patel, B.N.Suhagia, P.B.Shah, and R.R.Shah, Indian J. Pharma. Educ. and Research, 68, 635-638 (2006) L.J.Patel, B.N.Suhagia, and P.B.Shah, Indian Drugs, 43(8), 630-635 (2006) M.B.R Clarice, B.Liberato, F.Marico, D.M.Marcelo, B.Lisiane, and L.D.Sergio, J. Liq. Chromatogr. and Rel.Tech., 28, 477-486 (2005) PART II: Validation of analytical procedure: methodology Q2B, ICH Harmonized Tripartite Guidelines, 1996, p. 6-12.
Conclusion
The observed results evidenced that the proposed method can be used for simultaneous estimation of amlodipine besilate and bisoprolol fumarate in combined dosage form. The value within the limit of standard deviation and % relative standard deviation signifies high precision of method.
Acknowledgement
The authors would like to thank Head of Department. This study was supported by University Department of Pharmaceutical Sciences, RTM Nagpur University, Nagpur. Authors are also thankful to Ranbaxy Pharmaceutical Ltd. and Merck Pharmaceutical Ltd for gift samples of amlodipine besilate and bisoprolol fumarate respectively.
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Development And Validation of Spectrophotometric Method for Determination of Montelukast Sodium in Bulk and Tablet Formulation
Varun Pawar*, Lalitha N, Puranik SB, Sanjay Pai P.N, Rao G.K.
Abstract: Montelukast sodium, a specific cysteinyl leukotriene receptor antagonist, belongs to a styrylquinolines series and used in the treatment of asthma. A simple, rapid, precise and accurate spectrophotometric method has been developed for estimation of montelukast sodium in bulk and tablet formulation. In the proposed method water was used as solvent and wavelength maxima was selected at 283 nm. The proposed method was validated as per ICH method validation guidelines. Linearity was observed in the concentration range of 1 - 80 gmL-1 (r2= 0.9990). The assay results were found to be in good agreement with label claim. The recovery studies were carried out at three different levels i.e. at 80%, 100%, 120%. The method was validated statistically for LOD, LOQ, repeatability by precision study and accuracy by recovery studies.
Introduction
Montelukast sodium, a specific cysteinyl leukotriene receptor antagonist, belongs to a styrylquinolines series with the chemical name 2-[1-[1(R)-[3-[2(E)-(7-chloroquinolin-2-yl)vinyl] phenyl]3[2-(1-hydroxy-1-methylethyl)phenyl]propylsulfanylmethyl] cyclopropyl] acetic acid sodium salt used in the treatment of asthma (Fig. 1). Literature survey reveals that spectroflurometric, voltametric, and chromatotographic methods for estimation of montelukast in bulk and pharmaceutical formulations and biological fluids are available. The present study describes a simple rapid and economical UV-visible spectrophotometric estimation of montelukast in bulk and tablets. The method was validated as per ICH method validation guidelines.
Fig. 1: Structural formula of Montelukast
HO O
Materials and Methods Instrument and Material

Pure Montelukast sodium was obtained as gift sample from Sun Pharmaceuticals Pvt. Ltd. India. The spectrophotometer used was Shimadzu UV-Visible spectrophometer 1601 model with spectral bandwidth 3 nm and wavelength accuracy (with automatic wavelength correction) 0.5 nm. Analytical grade water was purchased from Qualigens Fine chemicals Mumbai, India. All the apparatus and instruments were calibrated and validated as per calibration and validation protocol before starting the experimental work.
Determination of wavelength maxima and molar absorptivity

Standard solution of concentration 50 gmL-1 of montelukast was prepared in 10 mL volumetric flasks using water as solvent and scanned in the UV range of 220 to 400 nm (Fig 2). The wavelength maxima of montelukast was found to be 283 nm. The absorbance of drug was recorded at 283 nm and molar absorptivity (C) for both the drugs was calculated from the formula: C = A/C.........................Eqn.1, Where, A: absorbance, C: concentration of analyte in g100mL-1.
Cl
Na
H3C HO CH3
The absorptivity of montelukast at 283 nm was found to be 383.15.
Al-Ameen College of Pharmacy, Near Lalbagh Main Gate, Bangalore. Author for correspondence E-mail: varunrpawar@yahoo.co.in
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Fig. 2: The UV-Spectra indicating wavelength maxima of Montelukast 3.00A Table 1: Summary of Validation Parameters SI. No. 1 2 (0.500) /div) 3. Precision LOD LOQ Linearity Parameters 2.53 7.66 gmL-1 Regression eq R
2 n
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Montelukast
10 - 80 0.0389x - 0.0129 0.9996 0.0488 0.101 0.0018171 0.101 0.0727 0.15 0.1112 0.2301 0.00026 98.3 % 98.34 % 98.7 %
Method (SD) %RSD System (SD) %RSD Inter-day (SD) %RSD
0.00A 220.0 Paax 283.2 1.725 (20/div) 320.0 4. 5. Sensitivity (g/cm3/AU) Accuracy
Intra-day (SD) %RSD 283 nm 80% 100% 120%
Preparation of standard stock solution

Standard stock solution was prepared by dissolving 10 mg of montelukast in water in 10 mL volumetric flask and volume was made upto 10 ml with water. Different aliquots were taken from the stock solution to obtain series of concentrations to get concentration in the range of 1-100 gmL-1. The absorbance of solution was recorded at the gmax 283 nm against water as blank.
deviation. In the present study, developed method was validated method, system, inter-day and intra-day precision. As the values of %RSD of all precision study were within the acceptable limits (less than 2%), both the method as well as the system provides good precision and reproducibility (Table 1). 4. Sensitivity Absorbance of standard solutions of montelukast was taken at 283 nm. Sandell's sensitivity (II) for the drug was calculated from formula, Conc. of drug (g100mL-1) X 0.001 II (g/cm3/AU) = Absorbance The Sandell's sensitivity for montelukast was found to be 0.00026 gcm -3AU-1. 5. Accuracy Accuracy of the method by was determined in the terms of % recovery of standard. Recovery studies were carried out by addition of standard drug solution at the level of 80%, 100% and 120% to the preanalyzed sample. Results of the recovery study were found to be within the acceptance criteria 10010 % and presented in Table 1.
Validation of Analytical Method

Validation of an analytical method is the process to establish that the performance characteristics of the developed method meet the requirements of the intended analytical application. 1. Limit of Quantification (LOQ) and Limit of Detection (LOD) LOD and LOQ were calculated statistically from formula, LOD = 3.3 (SD / S), LOQ = 10 (SD / S) Where, SD: Standard deviation of y- intercepts of regression lines S: Slope of the calibration curve The LOD and LOQ of montelukast by statistical method were mentioned in Table 1. 2. Linearity and range The linearity range of montelukast was found to be 1-80 gmL-1. The values of percentage curve fittings were in the limits of acceptance criteria (Table 1). 3. Precision Precision is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample. Precision is usually expressed as the standard deviation or relative standard
Procedure for the analysis of tablet formulation

Sample: Montelukast sodium Tablet Brand name: Montair 10 (10mg of Montelukast sodium). Manufacturer: CIPLA Ltd.
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ANALYTICAL METHOD DEVELOPMENT Preparation of sample stock solution

Accurately weighed and powdered 20 tablets. 189.3 mg of tablet powder equivalent to 10 mg of montelukast was weighed into a 100 mL volumetric flask. The powder dissolved in sufficient volume of water and filtered through a Whatmann filter paper. The volume of filtrate was made up to 100 mL with water. Volume of 5 mL was transferred to 10 mL volumetric flasks and volume was made up to 10 mL with water. Absorbance of this solution at 283 nm was recorded. Using regression equation, the concentration of montelukast in tablets was calculated. The amount of montelukast in marketed tablet formulation confirmed to the label claim (montelukast sodium 10 mg) (Table 2).
Table 2: Results of Assay Label Claim (mg/tablet) Montelukast (10 mg) Amount found* % Amount found 9.865 98.65 %RSD 0.243
7. 6.
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can be employed in the routine analysis in quality control laboratories.
References
1. 2. 3. 4. Beckette AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4th ed. The Anthlone Press; London; 1988: P. 157, 281-307. Montelukast (sodium salt), Catalog No. 10008318. Cayman Chemical. http/www.catmanchem.com. SOP:Validation studies. Indian Pharma Guidance Academy; Nagpur. 1996. P.1- 3. Alsara, Al-Omar, Gadkariem EA, Belal F. Voltametric determination of montelukast sodium in dosage forms and human plasma. II Farmaco 2005; 60(6):563-567. Alsarra I, Khalil NY, Sultan M, Al-Ashban R, Belal F. Spectrofluorometric determination of montelukast in dosage forms and spiked human plasma. Phaemazie 2005; 60(11):823-826. Radhakrishna T, Narasaraju A, Ramkrishna M, Satyanarayana A. Simultaneous estimation of montelukast and loratidine by HPLC and derivative spectroscopy methods. J Pharm. Biomed. Anal. 2003; 31(2):359-368. Al-Rawithi S, Al-Gazlan S, Al-Ahmadi W, Alshowaier IA, Yusuf A, Raines DA. Expedient liquid chromatographic method with fluorescence detection for montelukast in micro-samples of plasma. J Pharm. Biomed. Anal. 2001; 754(2): 527-531. Robert P, Pauline L, Wayne MM, Elizabeth K. A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry. J Chromatogr. B 2007; 858(1-2): 282-286.
5.
*Average of six determinations
Results and discussion

The proposed method for simultaneous estimation of montelukast and bambuterol was found to be simple, accurate, economical and rapid. The values of R2, %RSD and recovery study were within the limits of acceptance criteria indicate repeatability, reproducibility and accuracy of the method. Hence it
8.
Source: The Pharma Review, October 2008
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Gas Chromatographic Determination of Residual Solvents in Pharmaceutical Excipients

S. B. Puranik*, Varun R. Pawar, N. Lalitha, P. N. Sanjay Pai, G. K. Rao
Abstract: A systematic approach for the identification and quantification of organic volatile impurities (OVIs) in pharmaceutical excipients was described in the proposed Analytical method was developed utilizing capillary gas chromatography with flame ionization detector and validated as per ICH guidelines for residual solvent analysis in different pharmaceutical excipients. The separation was carried out on BP 624 column (30m X 0.53mm i.d. X 0.25m coating thickness), using GC 17 A shimadzu, with nitrogen as carrier gas in the split mode by direct injection method. The method described was simple, sensitive, rugged, reliable and reproducible for the quantitation of organic volatile impurities (OVIs) at residual levels as per ICH guidelines from pharmaceutical excipients.
Introduction
There is a growing appreciation of the role that pharmaceutical excipients play in enhancing stability, bioavailability, and delivery of active drugs. Purity of excipients is becoming increasingly important in the development and manufacture of pharmaceutical products as the quality of excipients can have a significant impact on the safety and efficacy of drug products.1-2 Organic solvents, which are widely used in the production of excipients for synthesis and purification, may not be completely removed by practical manufacturing techniques. The residue of these solvents offers no therapeutic benefit, but can present a serious potential hazard to human health. Therefore, it is essential to ensure that the residual solvents, (organic volatile impurities (OVIs)) should be below the acceptable levels stipulated in worldwide regulatory standards, such as ICH3 and USP4. The use of some highly toxic/carcinogenic solvents has to be controlled to extremely low levels and demands extremely sensitive detection methods. Analysis of OVIs is known to be one of the most challenging analytical tasks in pharmaceutical analysis and control. Excipients present an even greater challenge due to the fact that information regarding the presence of OVIs in excipients may be difficult to obtain from a supplier who is unwilling to provide comprehensive data on the materials because of fear that the information could be exploited by a competitor. Unknown OVIs are often detected during the routine quality control testing of OVIs in excipients using official methods, resulting in lengthy laboratory investigations which can cause costly delays in manufacturing. Hence, the situation necessitates
the development of a rapid, sensitive, and reliable analytical method to screen, identify, and quantitate any OVIs in pharmaceutical excipients. The most appropriate method for analyzing organic volatile compounds is capillary gas chromatography (cGC) due to its unique features, which include extremely high separation efficiency, the availability of a sensitive universal flame ionization detector (FID) system for quantification.
Experimental Method
1. Instruments and Materials Gas Chromatograph Shimadzu 17A version 3 was used in the development and validation of GC method. Gas chromatograph was equipped with standard oven for temperature ramping, split/splitless injection ports and flame ionisation detector. BP 624 column (30m X 0.53mm i.d. X 0.25m coating thickness, 4% cyanopropyl phenyl and 96% dimethyl polysiloxane stationary phase), with nitrogen as carrier gas in the split mode by direct injection method was used. Analytical grade solvents acetone, ethyl acetate, isopropyl alcohol, methanol, formaldehyde, toluene and dimethyl sulphoxide (DMSO) were purchased from Thomas Baker, Mumbai, India. Pharmaceutical excipients have been procured from different manufacturers. 2. Preparation of standard Dimethyl sulphoxide (DMSO) was selected as the standard and sample diluent, based on its ability to dissolve wide variety of substances and high boiling point that does not interfere with more volatile solvents analyzed by GC. Standard stock of each
AL-Ameen College of Pharmacy, Near Lalbagh Main Gate, Bangalore. *Author for correspondence E-mail: sangpur@rediffmail.com
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solvent acetone, ethyl acetatel, isopropyl alcohol, methanol, formaldehyde and toluene was prepared by diluting with DMSO. Working standard of each solvent ranging from concentration 100ppb to 5600 ppm was prepared with DMSO in 10 mL volumetric flasks. 1L of each working standard was injected in to gas chromatograph and standard calibration curve was obtained for each solvent acetone, ethyl acetate, isopropyl alcohol, methanol, formaldehyde and toluene. 3. Preparation of mixed standard Accurately weighed 1g sample of each pharmaceutical excipient dissolved with DMSO in separate 10 ml volumetric flask and filtered through whatman filter paper No 1.The volume was made up to 10mL with DMSO. From each sample 1L sample was injected into chromatograph and chromatogram was recorded. Concentrations of solvents viz; acetone, ethyl acetate, isopropyl alcohol (IPA), methanol, formaldehyde and toluene in all samples were calculated. 4. Gas chromatographic conditions The volume of 1 L standard and sample solution was injected in GC injection port. The temperature of injection port was maintained at 2300C, split ratio 1:15, with nitrogen as a carrier gas. The pressure of 14 kpa with flow of 3.2 mL min-1 was maintained. The temperature of the detector was set at 250OC. Temperature gradient maintained at 40OC for five min and then increased at a O -1 -1 rate of 10 Cmin to 550C min and maintained for 5min, finally increased at the rate of 10OCmin-1 to reach the final temperature of 200OC and maintained for 5 min.
Table: 1
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Excipients Manitol
Major use Filler
Methanol Acetone IPA ---200ppm ---------10ppm ------------------
Ethyl acetate Toluene Formaldehyde ---------1500 ppm -----------0.4 ppm 0.2 ppm 86 ppm
Tromethamine Alkalizing agent Lactose Polyethylene glycol 400 Carbomer Filler Solvent
Emulsifying --/suspending agent
Propyl gallate Antioxidant ---
---
80ppm ---
----
The same method was applied for the residual solvent analysis of manitol, tromethamine, lactose, polyethylene glycol-400, carbomer, propyl gallate, hydroxyl propyl methylcellulose, sodium dodecyl sulfate and polysorbates. Manitol showed presence of formaldehyde at the level of 0.4 ppm, tromethamine showed the presence of methanol and acetone at the levels 200 and 10 ppm respectively and lactose showed the presence formaldehyde at the level of 0.2 ppm, polyethylene glycol 400 showed the presence of formaldehyde at the level 86 ppm, carbomer showed the presence of ethyl acetate at the level 1500 ppm, propyl gallate showed the presence of IPA at the level 80 ppm, hydroxyl propyl methyl cellulose showed the presence of formaldehyde at the level of 16 ppm all were in the range of acceptable criteria. 2. Validation of method 2.1 Specificity The specificity of analytical method was determined by injecting the solvents used in the process, other than methanol and isopropyl alcohol. A blank solution of dimethyl sulphoxide was injected under the same experimental conditions. No peak was observed between 2 to 20 min in the chromatogram, only the peak of DMSO was observed at the end of chromatogram. 2.2 Precision Precision of the method and system expressed in terms of standard deviation and relative standard deviation. For the precision of method and system the %RSD for six solvents complies with the acceptance criteria of less than 2% (Table2), hence the method and system is said to be prcised. 2.3. Linearity All six solvents showed broad range of linearity (Fig.2). The correlation coefficient calculated for all six solvents lies in the acceptance criteria of more than 0.99 (Table 2). 2.4. Limit of Detection (LOD) and Limit of Quantitation (LOQ) The LOD and LOQ were calculated by instrumental and statistical
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Method Validation
The analytical method validation was carried out as per ICH method validation guidelines [9]. The validation parameters addressed were specificity, precision, linearity, limit of detection, limit of quantitation, ruggedness and system suitability.

1. Development of method Gas chromatographic method for the determination of residual solvents in pharmaceutical excipients was developed. The column used was BP624 capillary column, with flow rate 3.2mLmin-1, linear velocity 22 cmsec-1 and column pressure 14 kpa with total flow of 116 mLmin-1 in the split mode. In the prescribed method all six solvents will get eluted within 16 min.
22

Table: 2 Name of Solvents Range (ppm) Methanol 10-3200 Acetone 10-5600 Isopropyl 10-5600 alcohol Ethyl acetate
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Linearity Slope R2 SD
Precision System Method %RSD SD 1.35 2.25 8.94 0.61 2.39
LOD (ppm)
LOQ (ppm)
Ruggedness
%RSD Instru- Stat. Instru- Stat. Intra- Intermental mental day day 70 100 200 42.5 100 71.0 300 270 300 128 94.65 93.67
972.42 0.9997 1253.31 2108.8 0.9952 4543.16 1198.8 0.9971 9833.57
1.27 1.35 2.08 2.25 7.96 8.94 1.58 1.37 2.29 2.39 1.09 10.8
The values for the Limit of Detection and Limit of Quantification for are mentioned in Table 2.
215 92.12 89.57 2.5. Ruggedness 820 89.03 92.73 The ruggedness of
100-5600 670.35 0.9963 1050.26 3228.1 0.9995 7459.72 10170
Toluene 10-1600 Forma- 10-5600 ldehyde Figure: 1
0.9952 11547.01 10.84
3500000 3000000 2500000
Lineareity of Methanol y = 979.69x - 16185 R2=0.9998
method was 70 51.7 100 156 115.7 105.6 e s t a b l i s h e d b y performing same 80 76.2 150 231 95.66 95.87 c h r o m a t o g r a p h i c 60 37.4 100 113 100.7 95.45 system and the same column by two analysts on a different day. The assay results of ruggedness studies were in the range of 90-105% v/v (Table 2). Additionally, good separation between the peaks of standard was achieved, which indicated that the method was selective for all components under the test. 2.6. System suitability A system suitability tests viz number of theoretical plates, HETP, asymmetry and resolution was performed to evaluate the chromatographic parameters. The system suitability parameters calculated for all six solvents found in the acceptable range, which indicates the efficiency of the method.
Area
2000000 1500000 1000000 500000 0 -500000 0 500 1000 1500 2000 2500 3000 3500
Concentration
Conclusion
This study presents a simple and validated Gas Chromatographic method for estimation of residual solvents in pharmaceutical excipients. The developed method is specific, accurate, precise and rugged. The amounts of organic volatile impurities present in the pharmaceutical excipients were found to be within the ICH limits.
Linearity of Ethanol
70000000 60000000 50000000
y = 10386x - 843897 R2=0.996
Area
40000000 30000000 20000000 10000000 0 -10000000 0 1000 2000 3000 4000 Concentration 5000 6000
Acknowledgement
Highly thankful to Prof. B.G.Shivananda, Principal Al-Ameen College of Pharmacy, Bangalore and to all my research colleagues for their support in the work.
References
1. 2. 3. 4. 5. 6. 7. 8. Impurities in New drug Substances (1997) International Conference on Harmonization. Available from: http://www.ich,org. S. B. Puranik, P. N. S. Pai, G. K. Rao, IJPS. 69 (2007) 353. S. B. Puranik, Varun Pawar, Lalitha N, P. N. S. Pai, G. K. Rao, The Pharma Review. 6 (2008) 121. T. K. Chen, W. Moeckel, H. L. Superhant, Pharmaeuropa. 4 (1992) 62. Z. Li, Y. H. Han, G. Zhuang, J. Pharm. Biomed. Anal. 28 (2002) 673. C. C. Camarasu, J. Pharm. Biomed. Anal. 23 (2000) 197. S. C. Rastogi, Chromatographia 37 (1993) 211. M. A. Barrio, J Hu, P.Zhou, N.Cauchon, J. Pharm. Biomed. Anal. 41 (2005) 738.
methods. For the instrumental method LOD is determined as the lowest amount to detect and LOQ is the lowest amount to quantify by the detector. For statistical method LOD and LOQ determined by statistical formula. LOD = 3.3 SD/S LOD = 10 SD/S Where, SD is standard deviation of y intercept of linear regression and S is slope.

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UV-Spectrophotometry and First Order Derivative Methods for Estimation of Efavirenz in Bulk and Capsules
Charushila H. Bhirud, Atul A. Shirkhedkar*, Ravindra A. Fursule and Sanjay J. Surana
Abstract: Two simple, rapid, accurate and economical 'UV Spectrophotometry' and 'First order derivative' methods have been developed for determination of efavirenz in bulk and capsules. In acetonitrile, the gmax of the drug was found to be 247 nm. The same spectrum was derivatised into first order derivative using UV probe software of instrument (Shimadzu- 2450), at Dg = 2. The amplitude of the trough was recorded at 258 nm. Efavirenz follows linearity in the concentration range 4 - 24 g/ml in both these methods with correlation coefficient 0.9999 and 0.9998. The assay results were in good agreement with label claim. The methods were validated statistically and by recovery studies. The relative standard deviation were found to be less than 2% with excellent precision and accuracy.
Introduction
Efavirenz, (S) 6 chloro ( cyclopropylethynyl )- 1,4dihydro -4(trifluromethyl )- 2H- 3,1 benzoxazin2 - one is nonnucleotide reverse transcriptase inhibitor and used in the treatment of immunodeficiency virus.1,2 In literature, few chromatographic3-5 methods are reported for estimation of efavirenz from human plasma. The objective of the present work is to develop simple, rapid and economical 'UV-spectrophotometric' and 'first order derivative' methods for determination of efavirenz in bulk and finished products.
Fig : An overlain first order derivative spectra of efavirenz in acetonintrile
Material and Methods

Reagents All reagents were of analytical reagent grade. Preparation of standard stock solution and study of calibration curves Standard stock solution containing 100 g/ml efavirenz was prepared in acetonitrile. Different aliquots were taken from the stock, diluted to 10 ml mark with double reverse osmosis (R.O) water to obtain a series of concentrations. The solutions were scanned on UV- visible spectrophotometer (Shimadzu-2450 with UV probe 2.21 software) in the UV range 200 - 400 nm. Efavirenz showed absorbance maxima at 247 nm. The same spectra were derivatised into first order derivative, using UV probe software of instrument, where Dg = 4 (fig). The amplitude of the corresponding troughs were measured at 258
R.C. Patel College of Pharmacy, Karwand Naka, Shirpur, Dhule. *Address for Correspondence Email: atul_shirkhedkar@yahoo.com
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Abs
24

nm. In both the methods, efavirenz follows linearity in the concentration range of 4 24 g/ml. Preparation of Sample Solution To determine the concentration of efaverinz in capsule; contents of twenty capsules were weighed, their mean weight determined and finely powdered. The powder equivalent to 100 mg of efavirenz was transferred into 100 ml volumetric flask containing 30 ml acetonitrile, shaken manually for 10 min, volume was adjusted to mark with same solvent and filtered through Whatmann filter paper no. 41. An appropriate aliquot was transferred to 10 ml volumetric flask, volume was adjusted to the mark with double R.O. water and absorbance was recorded at 247 nm. The same spectrum was derivatised, using UV-probe software and amplitude of the trough was recorded at 258 nm. The results are shown in table 1
Table 1: Results of Assay UV-Spectrophotometry Drug Efavirenz (200 mg /capsule) Amount found (%) [ n = 6] 99.07 First order derivative %RSD Amount found (%) [ n = 6] 0.142 100.72 %RSD 0.601 Table 2: Summary of Validation Parameters Parameters % Recovery [n = 9] % RSD Precision [%RSD] Intra-day (n = 3) Inter-day ( n = 3) Repeatability (n = 6) Ruggedness [%RSD] Analyst-I (n =3) Analyst II (n =3) 0.723 0.729 0.737 - 0.953 0.493 - 0.821 0.675
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UV-Spectrophotometry First order derivative 99.69 0.401 100.14 0.241 0.505 - 0.890 0.385 - 0.517 0.820 0.702 0.703
spectrophotometry' and Y = 0.0038 X + 0.0012 (r2 = 0.9998) in 'first order derivative method'. Amount of drug estimated was in good agreement with the label claim. The methods were validated for accuracy, precision and ruggedness as per USP;6 the results are as shown in table 2. The results of validation parameters demonstrated that the procedure is accurate, precise and reproducible (relative standard deviation <2%), while being simple, economical, less time consuming and can suitably be applied for the determination of efaverinz from pharmaceutical formulation.
References
1. 2. 3. 4. The Merck Index, Maryadele J.O. Neil. Eds, In, 14th edn., Published by Merck and Co, White House Station, NJ, USA, 2006,598. Ren J, Bird LE, Chamberlain PP, et al. , Proc Natl Acad Sci ,2002,USA 99,14410 14415. A. Lakshmi Sailaja, K. Kishore Kumar, D. V. R. Ravi Kumar, C. Mohan Kumar, N. M. Yugandhar,G. Srinubabu, 2007, 65(5-6) , 359-361. Geetha Ramachandran, A.K. Hemanth Kumar, Soumya Swaminathan P.Venkatesan, V. Kumaraswami and David J. Greenblatt, J. Chromatogr. B, 2006, 835(1-2), 131- 135. Montgomery, E.R., Edmanson, A.L., Cook, S.C., Hovsepian, P.K, J. of Pharma. l and Bio.l Ana., 2001, 25(2), 267-284. United State Pharmacopeia, 28th edn., Rockville, MD, The United State Pharmacopoeial Convention, Inc: 2005, 2749-2751.
Recovery Studies The recovery studies were carried out at three different levels i.e. 80%, 100% and 120% level. To the preanalyzed sample solution, a known amount of standard drug solution was added and reanalyzed sample by the proposed methods. The results are shown in table 2.
Results and Disscussion

Efavirenz in acetonitrile showed absorbance maximum at 247 nm. In first order derivative spectrum, the amplitude of the trough was recorded at 258 nm. In both the methods, efavirenz follows linearity in the concentration range of 4 - 24 g/ml with linear regression equation Y = 0.0427 X + 0.0049 (r2 = 0.9999) in 'UV5. 6.
Source: The Pharma Review, June 2008
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RP-HPLC Estimation of Eplerenone in Tablets

K. P. Bhusari*, P. B. Khedekar, N. D. Amnerkar, S. M. Dhole, V. S. Banode
Abstract : A method for the determination of Eplerenone in tablets has been developed using reverse phase high performance liquid chromatographic method. The elution was done using a mobile phase consisting of methanol and water (75 : 25 v/v) on RPC18 250 x4.6 mm analytical column with flow rate of 1 ml/min with detection at 243 nm. The elution time was 4.02 min. The method was found suitable for quantitative estimation of eplerenone in tablets.
Introduction
Eplerenone (EPL) is chemically 9a,11a-Epoxy-7amethylcarboxy-3-oxo-17a-pregn-4-en-21,17b carbolactone, which is used as selective aldosterone bloker, antihypertensive and diuretic.1,2 Eplerenone binds to the mineralocorticoid receptor and blocks the binding of aldosterone, a component of reninangiotensin-aldosterone system.3,4 It is listed in Martindale.5 Literature survey revealed SPE LC-MS/MS assay for eplerenone and its hydrolysed metabolite in human urine.6 The present work describes the simple, isocratic RP-HPLC method for the determination of EPL in tablets. The developed method was validated using ICH guidelines for validation.7
Preparation of working standard solution: Working standard solution was prepared by diluting 1 ml of the stock solution to 10 ml with mobile phase (100 g/ml). Standard calibration curve: The various dilutions were prepared by taking 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 ml of solution and made up to 10 ml with mobile phase. Twenty microlitres of the solution from flask was injected. Calibration curve was constructed by plotting peak areas against the corresponding drug concentrations. The detector response was found to be linear in the concentration range of 10-200 g/ml. Estimation of drug in commercial tablet formulation: For the estimation of drugs from commercial formulation, twenty tablets each containing 25 mg of EPL were weighed and finely powdered. Accurately weighed tablet powder equivalent 50 mg of EPL was suspended in the mobile phase and shaken for 15 min. The volume was made up to 50 ml with mobile phase and filtered through whatman filter paper to produce 1000 g/ml. Aliquot portion of this solution was diluted to produce 100 g/ml and lilteved through membrane. Equal volume of 20 l of standard and sample solutions was injected separately after the equilibrium of stationary phase and area under the curve noted. Amount of the drug in the tablet formulation was calculated.
Materials and Method

The drug sample, Eplerenone, was obtained as gift sample from MSN Lab. Pvt. Ltd., Medak. HPLC grade methanol and water were supplied by Merck Co, Mumbai. Instrument: A liquid chromatographic system used Shimadzu Isocratic Liquid Chromatographic System model LC-10 AD, equipped with Rheodyne injector model 7725 with 20 l fixed loop, UV-Visible detector model SPD-10A, Mayura analytical Pvt. Ltd. The column C18 intersil (250 x 4.6 mm, i. d., particle size 5 ) was used as a stationary phase. Preparation of mobile phase: The mobile phase used was a mixture of methanol and water in the ratio of 75: 25 v/v and was filtered before use through whatman filter paper No.41. The elution was carried out at the flow rate of 1ml/min. Detection was carried out at 243 nm at ambient temperature. Preparation of solution: Preparation of standard stock solution : Standard stock solution of the drug was prepared by dissolving 50 mg of EPL in mobile phase and volume was made up to 50 ml with the same solvent (1000 g/ml).

In RP-HPLC method for estimation of EPL, mobile phase used was methanol and water in the ratio (75 : 25 v/v) at flow rate of 1 ml/min. The detector response was recorded at 243 nm. The retention time was found to be 4.02 min (Fig. 1). Linearity for EPL was in the range of 10-200 g/ml. To study the accuracy, reproducibility and precision of the proposed method, recovery experiments were carried out. A fixed amount of the preannalysed sample was taken and standard drug was added at three different levels. Each level was repeated atleast five times. The recovery values were in the range of 119.23% to 99.47%.
Sharad Pawar College of Pharmacy, Wanadongri, Nagpur. *Author for correspondence E-mail: nikhilamnerkar@gmail.com
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[mv] 150
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References
1. Cook C. S., Berry L. M. and Zhang L., Involvement of CYP3A in the metabolism of Eplerenone in humans and dogs: Differential metabolism by CYP3A4 and CYP3A5, Drug Met. Disp., 2002, 30(12), 1344-1351. Reyes A. J., Leary W, P, et. al., The aldosterone antagonist and facultative diuretic eplerenone: A critical review, Eur. J. Int. Med., 2005, 16(3), 145-153. Weinberger M. H., et. al., Effects of eplerenone versus losartan in patients with low-renin hypertension, Am. Heart J., 2005, 150(3), 426433. Pitt B., Remme W. and Zannad F., Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction, ACC Curr. J. Rev., 2003, 12(4), 57. Sweetman S. C., Eds., In; Martindale, The Complete Drug Reference, London: Pharmaceutical Press; Division of the Royal Pharmaceutical Society of Great Britain, 2007. (Electronic version. Available on http://www.medicinescomplete. com) Zhan J. N., Fast D. M. and Brean A. P., A validated SPELC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine, J. Pharm. Biomed. Anal., 2003, 31(1), 103. International Conference on Harmonization, Guidelines for Industry Q2A, Text on the Validation of Analytical Procedures, Q3A (R), Federal Register, 1996, 60 11260-11262.
Voltage
100
50
2.
0 0 2
3.
Retention Time (Min)
4 6 8 10
Fig. 1: A typical chromatogram of Eplerenone. For detailed chromatographic conclusions, see text.
4.
Conclusion
The present study comprises a RP-HPLC method to determine EPL from tablet dosage forms. The values of percent recovery and standard deviation indicate the method is accurate, reproducible and precise. The proposed method could be applied to routine analysis in quality control laboratories.
5.
6.
Acknowledgements
The authors are thankful to MSN Lab Pvt. Ltd., Medak for providing the drug sample of EPL for research work and also the Chairman, NYSS, Nagpur for providing necessary facilities and encouragement.
7.
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Development and Validation of New RP-HPLC Method with UV-Detection for the Determination of Carvedilol in Human Serum
Mogallapalli L V Setti, Vijaya Ratna J*
Abstract: Carvedilol is mainly used as an antihypertensive drug and it also possesses antioxidant and antiproliferative effects. A simple and sensitive analytical method was developed for carvedilol in human serum by using high performance liquid chromatography (HPLC). The method employs a liquid- liquid extraction for isolation and sample concentration followed by reversed phase liquid chromatography (RPLC) analysis using ultraviolet (UV) detection at 241 nm. Analytes were extracted from serum samples that were previously mixed with sodium hydroxide solution into an n- hexane, dichloromethane (7:3) solvent system. The mobile phase consisted of acetonitrile: orthophosphoric acid (37:63). The filtered mobile phase components were pumped from the respective reservoirs at a flow rate of 1.0 mL/min. Celecoxib was used as internal standard. Serum samples containing the carvedilol and internal standard, celecoxib were eluted through a C18 column. Retention times of carvedilol and celecoxib are 9.12 min and 11.49 min, respectively. The limit of detection of the drug in serum is 5 500 ng/ml. The extraction recovery of carvedilol is more than 75%. The intra day and inter day coefficient of variation and percent error values of the assay method were less than 5 %. Such a method would be ideally suitable for the estimation of carvedilol in pharmacokinetic studies after administration of carvedilol formulations.
Introduction
Carvedilol, (+)-1-(Carbaz ol-4-yloxy)-3-{[ 2-(omethoxyphenoxy)ethyl] amino}- 2-propanol (Figure 1), is a b receptor blocker and is an antihypertensive drug with a multiple action spectrum,1 and it also has vasodilating properties that are attributed mainly to its blocking activity at a1 receptors. Carvedilol is used in the treatment of mild to moderate hypertension and angina pectoris.2 Carvedilol undergoes extensive first pass metabolism that results in an absolute bioavailability of about 25%.3 Several techniques have been reported for carvedilol quantification in biological samples like plasma,4 serum,5 urine,6,7 and cardiac tissue.8 Literature survey reveals that complicated sample preparation and column switching procedure9 are the limitations in bioanalytical procedures. 7 Fluorescence measurement,4,10 and mass spectrometry are alternatives but they are very expensive procedures. The main objective of this study was to develop a simple, sensitive, reliable, economic, time
saving and fully validated HPLC method with UV detection, for the determination of carvedilol in human serum.
Experimental
Materials Carvedilol and celecoxib pure samples were gifted by Orchid Healthcare, Chennai and Torrent Pharmaceuticals, India, respectively. Acetonitrile and methanol (HPLC grade) were obtained from Rankem, India. Triethyl amine (AR grade) was obtained from SD fine chemicals, orthophosphoric acid (GR grade), dichloromethane (GR grade), and n-hexane (GR grade) were obtained from E-Merck, India. Triple distilled water was used during the entire HPLC procedure. Standard Solutions Primary stock solutions of 1 mg/mL of carvedilol and celecoxib were prepared in methanol. Appropriate dilutions of carvedilol from stock solution were made in methanol to produce working stock solutions of 5, 2, 1.5, 1, 0.5, 0.25, 0.1, and 0.05 g/mL.
AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam. *Author for correspondence Email: vijaya_ratna@yahoo.com
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These dilutions were used to spike serum in the preparation of a calibration curve. The internal standard (I.S.) working stock solution (2.5 g/mL) was made from the primary stock solution using methanol for dilution. Calibration samples were prepared by spiking 1 mL of individual blank serum with the appropriate amount of the drug on the day of the analysis. Samples for the determination of recovery, precision, and accuracy were prepared by spiking blank human serum with blanks of appropriate concentrations (5, 50, 100, and 500 ng/mL) of carvedilol. After preparation, the samples were stored at -20C till the analysis. Extraction Procedure To 1 mL of serum in a test tube, 100 L of carvedilol working solution and 100 L of celecoxib solution (2.5 g/mL) were added and the mixture was mixed well. To this, 300 L of 0.1 N sodium hydroxide solution was added and the mixture was vortexed for 1 min on a cyclomixer. Then, 5 mL of a solvent mixture (7:3 ratio of n-hexane: dichloromethane) was added to this solution and it was mixed for 5 min on a cyclomixer and was then centrifuged at 5000 rpm for 10 min. The organic phase was separated and the residue was subjected to re-extraction with 3 mL of the solvent mixture followed by centrifugation. The separated portions were pooled and subjected for evaporation in a vacuum oven. The residue was reconstituted in 1 mL of mobile phase and 20 L of this solution was spiked on to the HPLC column. Chromatographic Conditions A gradient HPLC (Shimadzu HPLC Class VP series, Shimadzu Corporation, Kyoto, Japan) with 2 LC-10AT VP pumps, a variable wavelength programmable UV/VIS Detector SPD-10A VP, a CTO-10AS VP column oven (Shimadzu), an SCL-10A VP system controller (Shimadzu), and a reversed-phase C18 column (250 mm 4.6 mm ID; particle size 5 m) (Shimadzu) were used. The HPLC system was equipped with the software Class-VP series version 5.03 (Shimadzu). The mobile phase consisted of acetonitrile, 15 mM orthophosphoric acid (37:63), and 0.25% v/v triethyl amine mixture, and was adjusted to pH 3.0 with 5% orthophosphoric acid. The filtered mobile phase components were pumped from the respective reservoirs at a flow rate of 1.0 mL/min. The column temperature was maintained at 30C. The eluent was detected by UV detector at 241 nm, and the sensitivity was set at 0.001 AUFS. Linearity, Limit of Detection, and Limit of Quantification The calibration samples were prepared by spiking 1 mL of blank human serum with the appropriate amounts of carvedilol and celecoxib on the day of analysis. The limit of detection (LOD) is
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the lowest level of the drug that can be detected in sample, but not necessarily determined in a quantitative fashion, using the specific method under the required experimental conditions. The limit of quantification (LOQ) was defined as the lowest concentration at which the RSD and deviation from the nominal concentration were less than 20%. Assay Validation The intra- and inter- run precision and accuracy of the assay (n=5) were determined by percent coefficient of variation and percent error values, respectively, based on reported guidelines11 like FDA guidelines. Samples containing 5, 50, 100, and 500 ng/mL concentrations were prepared for the determination of precision and accuracy. Samples were processed by appropriately spiking blank human serum, to get concentrations of 5, 50, 100, and 500 ng/mL. 1 mL of serum of each concentration was distributed into screw capped tubes and stored at -60C. Five replicates at each concentration were processed as described in the sample preparation, on day 1, 3, 5, and 10 to determine intra day and inter day precision and accuracy. The percent error values were calculated by the following equation.
Percent error = (observed concentration Added concentration) Added concentration X 100
Extraction Efficiency The efficiency of the extraction method to recover carvedilol and celecoxib from serum was tested using samples containing 5, 50, 100, and 500 ng/mL carvedilol and appropriate concentrations of celecoxib. These samples were then subjected to the sample preparation procedure explained above. The areas of carvedilol and celecoxib in the chromatogram were then compared with those of standard graph containing equivalent concentrations of carvedilol and celecoxib in the injected samples.

Chromatography The chromatograms of blank serum and serum sample having carvedilol and celecoxib (I.S.) are shown in Figures 2 and 3. In this analytical process, carvedilol and celecoxib were resolved with good symmetry and the retention times of carvedilol and celecoxib were 9.12 and 11.49 min, respectively (as shown in Figure 3). No endogenous interfering peaks were observed in the individual blank serum at the retention times of carvedilol and celecoxib, thereby, confirming the specificity of the analytical
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OH OCH2CHCH2NHCH2CH2O CH3O N H
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intercept. The equation of the calibration curve obtained from 6 points ranging from 5 500 ng/mL is y=0.0561x-0.4493 (as shown in figure 4). The correlation coefficient (r) between carvedilol concentration and ratio of peak areas of carvedilol and internal standard in human serum is 0.9995.
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RATIO OF AREAS
CALIBRATION CURVE FOR CARVEDILOL IN HUMAN SERUM
Figure 1: Structure of Carvedilol.

90000 70000 50000
mV
20 15 10 5 0 0
y = 0.0561x - 0.4493 r = 0.9995
100
200
300
400
500
SERUM CONENTRATION
30000 10000 -10000

2 4 6 8 10 12 14
Figure 4: calibration curve for carvedilol in human serum (celecoxib used as an internal standard) (n = 6)
(Time (min)
Figure 2: Typical HPLC chromatogram for analysis of blank serum.
110000 90000 70000

mV
50000
30000 10000 -10000

1 2 3 4 5 6 7 8 9 (Time (min) 10 11 12 13 14 15
Figure 3: Typical HPLC chromatogram of carvedilol. Retention time of carvedilol is 9.12 min; celecoxib is 11.49 min. (The representative concentration was 50 ng/mL of carvedilol)
method. Tailing factor was less than 1.1 for both carvedilol and celecoxib. The resolution between carvedilol and celecoxib was 3.6. Limit of Quantification The ratio of peak area of carvedilol to that of I.S was used for the quantification of carvedilol in serum samples. The calibration curves were linear in the concentration range 5-500 ng/mL. The type of regression equation is y = mx + c, where y represents the peak area ratio of carvedilol to celecoxib, x represents the concentrations of carvedilol, m is slope of the curve, and c is the
The LOQ, established by determining the concentrations of carvedilol in four spiked calibration standards having reproducibility with a relative standard deviation (RSD) less than 20% and an accuracy of 80 to 120%, was found to be 5 ng/mL and it was twice the value of LOD. The LOD was 2.5 ng/mL with RSD of 23%, and reproducibility was not observed with this concentration. The LOQ is observed to be approximately twice of the value of LOD. Using this method, it is possible to further increase the sensitivity by increasing the drug concentrations in serum. The intra day precision of the assay was determined by analyzing serum samples at each concentration on the same day. For the determination of inter day precision, the samples were analyzed on five different days. The intra day and inter day coefficient of variation (% CV) and percent error (%) values are shown in Table 1. These values are within the limits specified for inter and intra day precision.13 The recovery of carvedilol from serum was estimated at 5, 50, 100, and 500 ng/mL concentrations. Serum samples (in triplicate) containing carvedilol and celecoxib were extracted (1 mL serum with 10 mL of solvent mixture) and were analyzed. Triplicate samples containing similar concentrations of carvedilol in mobile phase were directly injected, and peak areas were measured. Absolute recovery was calculated by comparing the peak areas of pure samples spiked with the same amount of carvedilol and proceeded similarly. The absolute recoveries ranged from 7794% and are shown in Table 2. The accuracy of the method was verified by comparing the concentrations of carvedilol measured in extracted serum with the actual concentrations added.
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Table 1: Intraday and Inter day accuracy and precision of the assay (n = 5)
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References
1. 2. 3. 4. 5. Ruffolo, R.R., Jr.; Feuestein, G.Z . Cardiovasc. Drugs Ther. 1997; 11:247. Ruffole, R.R.; Gellai, M.; Heible, J.P.; Willette, R.N.; Nicholas, A.J. Eur. J. Clin. Pharmcol. 1990; 38:S82. Trefor, M. Clin. Pharmacokinet. 1994; 26(5):335. Hokama, N.; Hobara, N.; Kameya, H.; Ohshiro, S.; Sakanashi, M. J. Chromatogr. B 1999; 732:233. Clohs, L.; Mc Erlance, K.M. J. Pharm. Biomed. Anal. 2001; 24:545. Reiff, K. J. Chromatogr. 1987; 413:355. Gergov, M.; Robson, J.N.; Duchoslav, E.; Ojanpera, I. J. Mass Spectr. 2000; 35 (7):912. Behn, F.; Laer, S.; Seholz, H. J. Chromatogr. Sci. 2001; 39 (3):121. Lamrecht, G.; Staschitzky, K. Chromatographia 2004; 59:551.
Added Calculated conc. CV (%) Error (%) Conc. (ng/mL) (ng/mL) Intra day Inter day Intra day Inter day Intra day Inter day 5 50 100 500 5.16 50.60 100.72 500.80 5.18 50.31 100.69 499.98 3.92 3.43 1.45 1.46 4.25 3.40 1.15 1.90 3.20 1.20 0.72 0.16 3.60 0.62 0.69 -0.004
Table 2: Recovery and accuracy of the proposed method Conc ng/mL 5 50 100 500 Absolute recovery (%) MeanSD Range (n = 3) (Min-Max) 77.121.87 87.161.60 91.242.10 94.312.70 74.58-78.45 86.80-88.90 88.10-93.40 92.52-97.05 Accuracy (%) MeanSD Range (n = 3) (Min-Max) 94.801.56 96.281.24 95.902.81 96.632.52 93.5-96.3 95.4-97.7 92.9-98.5 94.2-99.3
6. 7. 8. 9.
10. Ptacek, P.; Macek, J.; Klima, J. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; 789 (2):405. 11. Shah, V.P.; Midha, K.K.; Findlay, J.W.; Hill, H.M. Pharm. Res. 2000; 17:1551. 12. Quality Assurance of Pharmaceuticals. A Compendium of Guidelines and Related Materials; Pharma book Syndiacate: India, 2002; Vol. 1, 116 to 149.
Conclusions
These experiments confirm that the present method for the determination of carvedilol in human serum is simple, selective, sensitive, specific, accurate and precise. The calibration curve is linear in the concentration range of 5 - 500 ng/mL. Hence such a method would be ideally suitable for the estimation of carvedilol in pharmacokinetic studies.
13. Bressolle, F.; Bromet, M.; Audran, M. J. Chromatogr. B 1996; 686:3.
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Gas Chromatographic Determination of Methanol and Isopropyl Alcohol Impurities in Herbal Extracts
S. B. Puranik*, Varun R. Pawar, Lalitha N, P. N. Sanjay Pai, G. K.Rao
Abstract: Residual solvents in herbal extracts were monitored using gas chromatography (GC) with Flame Ionisation detector (FID). As per GMP, measuring residual solvents is mandatory. It is now possible to take advantage of GC equipment with faster temperature ramping capabilities, in combination with shorter capillary GC columns, to achieve considerable gain in efficiency and reduction in analysis time. In the present study Gas chromatographic method for the determination of methanol and isopropyl alcohol at residual levels in herbal extracts was developed using a flame ionization detector and the separation was carried out on BP 624 column (30m X 0.53mm i.d. X 0.25m coating thickness), using GC 17 A shimadzu, with nitrogen as carrier gas in the split mode by direct injection method. The retention time for standard methanol and isopropyl alcohol was found to be 3.095 and 4.097 respectively. The linearity for methanol and Isopropyl alcohol was found to be in the range of 10-400 LmL-1 and 1-240 LmL-1 respectively. The method was validated according to ICH guidelines. The method described is simple, sensitive, rugged, reliable and reproducible for the quantitation of methanol and isopropyl levels from herbal extracts and their levels are found to be within the ICH limits.
Introduction
Herbal extract is a liquid extract of herbs, dried or fresh herbs are combined with solvent, and then the solid matter is removed leaving only the required active constituents of the herbs mixed in the solvent. Herbal extracts are sold as dietary supplements and alternative medicine. The active constituents in the herbs are extracted by using various solvents methanol, isopropyl alcohol, acetone, toluene, butanol, dichloromethane etc. These solvents cannot be completely removed by practical processes such as freeze drying and drying at higher temperature under vaccum. The fraction of solvents always remains with the extract and are referred as residual solvents or organic volatile impurities. The residual solvents have no therapeutic benefits and are toxic and hazardous to human health. ICH has prescribed the limits for the solvents in herbal extracts and formulations.2 The content of residual solvents in herbal extracts is routinely measured by gas chromatography. Routine GC applications include analysis of herbal extracts to comply to good laboratory and good manufacturing practices as well as in process testing of residual solvents to optimize drying procedure.3 Over the last decade, several GC methods to monitor residual solvents have been reported in the literature.4,5,6,7,8 For the said purpose, method has been developed and validated for detection and quantification of residual solvents methanol, isopropyl alcohol in herbal extracts of
1
Momardica charentia, Morinda Citrifolia, Rosemary, Curcumin, Green tea, Gymnema, Phyllantus niruri.
Experimental Method
1. Instruments and materials Gas Chromatograph Shimadzu 17A version 3 was used in the development and validation of GC method. Gas chromatograph was equipped with standard oven option for temperature ramping, split/splitless injection ports and flame ionisation detector. BP 624 column (30m X 0.53mm i.d. X 0.25m coating thickness, 4% cynopropyl phenyl and 94% dimethyl poysiloxane stationary phase), with nitrogen as carrier gas in the split mode by direct injection method was used. Analytical grade solvents methanol, isopropyl alcohol and dimethyl sulphoxide (DMSO) were purchased from Thomas Baker, Mumbai, India. Sample herbal extracts of Momardica charentia, Morinda Citrifolia, Rosemary, Curcumin, Green tea, Gymnema, Phyllantus niruri were obtained as gift samples from Phytotech Herbal Extracts Pvt Ltd. Bangalore, India. 2. Preparation of standard Dimethyl sulphoxide (DMSO) was selected as the standard and sample diluent, based on its ability to dissolve wide variety of substances. Also DMSO is a solvent with high boiling point that does not interfere with more volatile solvents tested by GC for the
Al-Ameen College of Pharmacy, Near Lalbagh Main Gate, Bangalore. Author for correspondence: Email: sangpur@rediffmail.com
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method involving analysis of high boiling point solvents. Standard stock of methanol and isopropyl alcohol were prepared by diluting with DMSO in 10 ml volumetric flask to get concentration of 1000LmL-1. From these stocks 8 serial working standard solutions were prepared to obtain concentrations ranging from 10-400LmL-1 and 1-240 LmL-1 for methanol and isopropyl alcohol respectively, volume is made with DMSO. 1L of working standards were injected in to gas chromatograph and standard calibration curves were obtained for methanol and isopropyl alcohol. 3. Preparation of sample Accurately weighed 1g sample extracts of Momardica charentia, Morinda Citrifolia, Rosemary, Curcumin, Green tea, Gymnema, Phyllantus niruri dissolved and sonicated with DMSO, filtered through whatman filter paper No 1 and volume made up to 10mL with DMSO, in separate 10mL volumetric flask. From these samples 1L samples were injected and concentrations of methanol and isopropyl alcohol in sample were calculated by interpolating standard calibration curve. 4. Gas chromatographic conditions Following experimental conditions were used; 1 L volume of either standard or sample solutions was injected in GC injection port. The injection port maintained at temperature 2300C with a split ratio 1:15. Nitrogen used as a carrier gas with pressure 14 kpa for an expected flow of 3.2 mLmin-1. Temperature of the detector was set at 2500C. Temperature gradient maintained at 400C for five min and then increased at a rate of 100Cmin-1 to 550C and maintained for 5min, finally increased at the rate of 100Cmin-1 to reach the final temperature of 2000C and maintained for 5 min.
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and isopropyl alcohol residual levels in herbal extracts was developed. The column used was BP624 capillary column, with flow rate 3.2mLmin-1, linear velocity 22cmsec-1 and column pressure 14kpa with total flow of 116mLmin-1 in the split mode. The retention time for standard methanol and isopropyl alcohol was found to be 3.095 and 4.097 min respectively. The samples of Momardica charentia Morinda, Citrifolia, Rosemary, Curcumin, Green tea, Gymnema, Phyllantus niruri have shown presence of methanol and isopropyl alcohol in different concentrations (Table 1). Table 1: Amount of Residual Solvents in Herbal Extracts
Sl. Herbal Extracts No. 1 2 3 4 5 6 7 Morinda Citrifolia Rosemary Curcumin Green tea Gymnema Phyllantus niruri Methanol Isopropyl Alcohol Area Conc. Area Conc. (LmL-1) (LmL-1) 223707 1572283 1075352 2531963 528413 4185379 1140 55 790 540 1290 273 1720 1621 637608 5606 10240 4433 936298 2439 0.9 980 3.15 5.7 2.45 1410 1.4
Momardica charentia 118189
2.
Validation of method
2.1 Specificity The specificity of analytical method was determined by injecting a blank solution, pure dimethyl sulphoxide solution under the same experimental conditions. No peak was observed from the chromatogram obtained by injecting 1L of DMSO as a blank. 2.2 Precision The precision of method is the extent to which the individual test results of multiple injections of the series of standard agree. Precision of the analytical method usually expressed in standard deviation and relative standard deviation (coefficient of variance). Standard deviation and %RSD for methanol found to be 505.09, 0.2585 and for isopropyl alcohol 959.32, 0.6213 respectively. 2.3. Linearity The calibration curves were obtained in a concentration range
Fig. 2
Linearity of methanol
Method validation
The analytical method validation was carried out as per ICH method validation guidelines.9 The validation parameters addressed were specificity, precision, linearity, limit of detection, limit of quantitation, ruggedness and system suitability. Standard plots were constructed for methanol and isopropyl alcohol in the range of 10400 LmL-1 and 1-240 LmL-1 respectively. Precision of the instrument has been carried out for methanol and isopropyl alcohol of concentrations 200 LmL-1 and 120 LmL-1 respectively. From this stock five successive injections of 1L were injected, from the area recorded in the chromatograms of methanol and isopropyl alcohol standard deviation and relative standard deviation (%RSD) were calculated.

1. Development of method Gas chromatographic method for the determination of methanol
area
Concentration
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Fig. 3 Linearity of IPA
2000000 1500000 Area 1000000 500000 0 0 500 1000 1500 2000 2500 3000 y = 660.17x + 11104 R2= 0.9964
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impurities present in the herbal extracts were found to be within the ICH limits. The method could be applied with success even to the analysis of marketed herbal extracts.
Acknowledgement
Highly thankful for Phytotech Herbal Extracts Pvt Ltd. Bangalore, India. for providing the gift samples, and to all my research colleagues for their support in the work.
Concentration
References
1. P K Mukherjee (2002) Quality Control of Herbal Drugs, 1st Edition, Bussiness Horizons: New Delhi, p380. 2. Impurities in New drug Substances (1997) International Conference on Harmonization. Available from: http://www.ich,org. 3. S B Puranik, P N S Pai, G K Rao (2007) Organic Volatile Impurities in Pharmaceuticals. Indian Journal of Pharmaceutical Sciences 69: 353. 4. A M Diwedi (2002) Residual Solvent Analysis in Pharmaceuticals. Pharma. Technol. 26:42. 5. T K Chen, W Moeckel and H L Superhant (1992) Modifications of the Europiean Pharmacopoeias Proposal on Residual Solvents. Pharmaeuropa 4:62. 6. Z Li, Y H Han and G Zhuang (2002) Stastic Head Space Gas Chromatographic Analysis of Residual Solvents in Gel Extrusion Model Tablet Formulatios. J. Pharm. Biomed. Anal. 28:673. 7. C C Camarasu (2000) Head Space SPME Method Development for the Analysis of Volatile Polar Residual Solvents by GC-MS. J. Pharm. Biomed. Anal. 23:197. 8. S C Rastogi (1993) Gas Chromatographic Analysis of Organic solvents Mixtures and Capillary Columns of Different Polarity. Chromatographia 37:211. 9. Impurities in New drug Substances (2002) International Conference on Harmonization. Available from: http://www.ich,org. 10. D A Lambropoulou, I K Konstantinou and T A Albanis (2007) Recent developments in headspace microextraction techniques for the analysis of environmental contaminants in different matrices. J. Chromatogr. A 1150: 70. 11. A Kurganov (2007) Mass-balanced definition of corrected retention volume in gas chromatography. J. Chromatogr. A 1150: 100. 12. A lvaro, G Lopez, Vanesa, Esquiu, Mara, and T Teresa (2007) Comparison of three gas chromatography methods for the determination of slip agents in polyethylene films. J. Chromatogr A 1150: 178. 13. M Rocheleau, M Titley1 and J Bolduc (2004) Measuring residual solvents in pharmaceutical samples Using fast gas chromatography techniques. J. Chromatogr. B 805: 77. 14. United States Pharmacopoeia (USP-NF XXIV) (1995) Pharmacopoeial Convention Inc. Rockville, Maryland, United states, p1877. 15. H H Willard, L Merritt (1986) Instrumental Methods of Analysis, 7th Edition, CBS Publishers and Distributors. New Delhi, p540.
10400 LmL-1 and 1-240 LmL-1 for methanol and isopropyl alcohol respectively. For methanol twelve serial concentrations were performed and the calibration equation obtained was y = 1940.6x with R2 = 0.9978 (fig.2). For isopropyl alcohol nine serial concentrations were performed and the calibration equation obtained was y = 660.17x +11104 with R2 = 0.9964 (fig.3). 2.4. Limit of Detection (LOD) and limit of Quantitation (LOQ) The Limit of Detection for methanol and isopropyl alcohol was found to be 1LmL-1. Limit of Quantitation for methanol and isopropyl alcohol was found to be 2LmL.-1 2.5. Ruggedness The ruggedness was established by determining methanol and isopropyl alcohol using the same chromatographic system and the same column by two analysts on a different day. The assay result indicated that the method was capable with high precision. Additionally, good separations were achieved, which suggested that the method was selective for all components under the test. 2.6. System suitability A system suitability test was performed to evaluate the chromatographic parameters (number of theoretical plates, asymmetry of the peak) before the validation runs. Three replicate injections of the standard solution and three injections of the solution prepared for the specificity procedure were used. The retention time of methanol and isopropyl alcohol was found to be 3.095 and 4.097 min respectively. HETP and No. of theoretical plates for methanol found to be 2.26, 1326 and for isopropyl alcohol 1.29, 2324 respectively.
Conclusion
This study presents a simple and validated Gas Chromatographic method for estimation of residual solvents methanol and isopropyl alcohol in herbal extracts. The developed method is specific, accurate, precise and rugged. The amounts of organic volatile
Source: The Pharma Review, February 2008
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Simultaneous Spectrophotometric Estimation of Paracetamol and Tramadol Hydrochloride in Solid Dosage Form
P.G. Yeole, S.J. Wadher*, M.P. Puranik, R.O. Ganjiwale
Abstract: Two simple, accurate and reproducible spectrophotometric methods, requiring no prior separation have been developed for simultaneous estimation of tramadol and paracetamol in combined dosage form. The first method employs formation and solving of simultaneous equations using 217 nm and 248.6 nm as the two wavelengths for forming equation. The second method involved formation of isoabsorptive equation at 224.2 nm (isoabsorptive point) and at 248.6 nm as a wavelength of paracetamol using methanol as solvent. Both the drugs and their mixtures obey Beer-Lambert's law at selected wavelengths at given concentration range. The methods have been validated statistically and by recovery studies.
Introduction
Tramadol is an analgesic agent, not official in any pharmacopoeia, chemically it is Trans-()-2-{(dimethylamino) methyl}-1-(3methoxyphenyl) cyclohexanol hydrochloride. Literature survey revealed a spectrophotometric method1 for its estimation. Several capillary gas chromatographic2 and HPLC3, 4 methods have been reported for its estimation. Paracetamol is an antipyretic agent; chemically it is N-(4hydroxyphenyl) acetamide. It is officially in I.P.5, B. P.6 and U. S. P.7 The review of literature revealed that no method is as yet reported for the simultaneous estimation of both the drugs in combined dosage form. This paper describes two simple, rapid, accurate and reproducible methods for simultaneous determination of both the drugs in tablet dosage form.
the concentration range 0-35 g/ml of tramadol and paracetamol. All dilutions were scanned in the wavelength range of 400-200 nm. Fig-1 represents the overlain spectra of both the drugs. Two wavelengths selected for the formation of simultaneous equation are 248.6 nm and 217 nm ( max of paracetamol and tramadol). Molar absorptivity determined for tramadol at 248.6 nm and 217 nm are 338.09 and 20.03 while respective values for paracetamol were 214.12 and 925.14. These were the mean of five independent determinations. The simultaneous equations formed were: Cx = Cy = A2 x 214.12 - A1 x 925.14 308491.18 (1)
Experiment
Instrument A Shimadzu UV/ Visible spectrophotometer-2401 with UVPC soft wear and with 10 mm matched quartz cell. Reagent: Methanol All chemicals/solvents were used during project were of AR grade. All solvents were filtered using Whatman filter paper No. 41.
A1 x 20.03 - A2 x 338.09 (2) 308491.18 Where, A1 and A2 are absorbance of sample solution at 217 nm and 248.6 nm respectively. Cx and Cy are concentration (in gm/100 ml) of paracetamol and tramadol respectively in sample solution. By substituting the value of A1 and A2 in equation (1) the value of Cx can be obtained. Now by substituting the value of A1 and A2 in equation (2) the value of Cy can be obtained. The validity of formed equations was checked by preparing five mixed standard, measuring there absorbance at respective wavelength and comparing these with the concentration calculated using above formed equations. The results of validation of simultaneous equation are shown in Table 1.
Procedure Method 1: Employing Simultaneous Equations

Pure drug sample of tramadol and paracetamol were dissolved separately in methanol so as to give several dilutions of standard in
Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha. *Author for correspondence E-mail: sjwadher@rediffmail.com
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Table 1: Validation of simultaneous equation using mixed standard Sr. Concentration Absorbance % Concentration No. of drug in mg at found Tramadol Paracetamol 217 nm 248.6 nm Tramadol Paracetamol 1 2 3 4 5 99.40 100.0 99.50 100.6 100.0 100.5 99.20 101.7 102.0 100.8 0.548 0.545 0.551 0.557 0.554 0.955 0.928 0.957 0.963 0.961 98.65 99.03 99.30 99.60 99.43 100.50 98.95 99.60 99.92 100.91
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wavelengths, one being the isoabsorptive point of the two components and other being the wavelength of maximum absorption of one of the two components. From the overlain spectra of two drugs absorbance were measured at selected wavelength i.e. 224.2 nm isoabsorptive point and 248.6 nm, g max of paracetamol (Fig. 1). The concentration of each component can be calculated by mathematical treatment of mentioned equations. For paracetamol
Cx = Qm - Qy x Qx - Qy Qm - Qx x Qx - Qy A Ax1 A Ay1 (3)
Procedure for analysis of tablet formation

Twenty tablets were weighed accurately and average weight per tablet calculated. Tablets were ground to fine powder. A quantity equivalent to 100 mg of paracetamol was transferred to 100 ml volumetric flask. The tramadol present in this tablet powder was 11.38 mg which could not be found out accurately due to low absorbance at lower concentration due to lack of strongly absorbing chromophoric group in its structure9, hence to increase the accuracy, accurately weighed 88.62 mg pure drug sample of tramadol was transferred to the same volumetric flask, powder was dissolved in 50 ml methanol with vigorous shaking and volume was made to mark with methanol. The solution was further diluted to get final concentration of 10 g/ml of paracetamol (or tramadol). The absorbance of the solution was measured at 217 nm and 248.6 nm and concentration of the two drugs was calculated using equation (1) and (2). The results of tablet
Table 2: Results of analysis of commercial tablets by proposed methods Sample Label claim mg/tablet % of Label claim Estimated* Method I 1 37.50 325 *Mean of five determinations 99.86 99.31 Method II 99.73
For tramadol Cy =
(3)
Where, Qm= ratio of absorbance of mixture at 224.2 nm and 248.6 nm Qx= ratio of absorptivity of paracetamol at 224.2 nm and 248.6 nm Qy= ratio of absorptivity of tramadol at 224.2 nm and 248.6 nm A= absorbance of mixture at isoabsorptive point Ax1 and Ay1 absorptivity of paracetamol and tramadol at isoabsorptive point. Cx and Cy = Concentration in g/100 ml of paracetamol and tramadol respectively in final solution. The drug can be estimated by the formula Cxd % estimation of drugs = Where, C=Cx=Cy= Concentration in g/100 ml of paracetamol and tramadol d= dilution factor W= weight of drug either paracetamol or tramadol in mixture Results of validation of isoabsorption ratio method are shown in Table 3.
Table 3: Validation of isoabsorptive ratio method using mixed standard Sr. Concentration Absorbance % Concentration No. of drug in mg at found Tramadol Paracetamol 224.2 nm 248.6 nm Tramadol Paracetamol 1 2 3 4 99.20 101.0 99.70 102.0 100.0 100.0 100.50 100.0 101.4 101.2 0.609 0.624 0.612 0.627 0.620 0.934 0.957 0.928 0.961 0.955 99.61 101.24 100.70 99.81 100.14 98.81 100.73 98.13 98.86 99.33
100
Tramadol Paracetamol Tramadol Paracetamol Tramadol Paracetamol 99.74
formulation are shown in Table 2.
Method 2: Isoabsorptive Point Method

In this method the absorbance was measured at two
2.500 2.000 1.500 1.000 0.500 0.000 200.0 250.0 300.0 Wavelength (nm) 350.0 400.0
Fig.1: Overlain spectra of paracetamol and tramadol in method.

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ANALYTICAL METHOD DEVELOPMENT Procedure for analysis of tablet formulation

Tablet solution was prepared in methanol as described earlier and was further diluted to obtain the solution having concentration 10g/ml of paracetamol (or tramadol). This solution was then analyzed to obtain the spectra and absorbance value at 224.2 nm and 248.6 nm were noted. These values were then equated in above mention equation (3) and (4) and the concentration of each drug was calculated.
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determination of absorbance of sample at the two selected wavelengths and few simple calculations. The second method employed the graphic absorbance ratio at two selected wavelength, once the absorbance is determined. The reported methods were found to be accurate, simple and rapid. Hence it can be employed for routine analysis of both drugs in quality control laboratories.
Recovery Studies
To study the accuracy, reproducibility and precision of the above proposed methods, recovery studies were carried out by addition of standard drug to pre-analyzed sample. Results of recovery studies were found to be satisfactory and reported in Table 4.
Table 4: Recovery study data Sr. Concentration of added Amount recovered (mg) No. amount of drug in mg 1 2 3 87.0 84.0 87.20 5.0 10.0 15.0 86.70 82.61 86.43 4.91 9.97 15.04 % Recovery
Acknowledgement
Authors are grateful to Modi-MundiPharma Pvt. Ltd, for gift sample.
References
1. H. E. Abdellataf, Kinetic spectrophotometric determination of tramadol in pharmaceutical formulation, J. Pharm. Biomed. Anal., 31; 29 (5), 835 (2002). R. Becker and W. Lintz, Determination of tramadol by capillary gas chromarography with nitrogen selective detection, J. Chromatogr., V377, 213 (1986). J. Duan, X. D. Sun and Z. X. Wang,, Rp-HPLC assay for plasma concentration of tramadol hydrochloride and Evaluation of its pharmacokinetics, Chinese J. Clinical Pharmacy, V6, 58 (1997).
2. 99.65 98.35 99.11 98.58 99.70 100.26 3.
Tramadol Paracetamol Tramadol Paracetamol Tramadol Paracetamol

The value of standard deviation was satisfactorily low and recovery was close to 100 % include the reproducibility and accuracy of both the methods. The first method employing simultaneous equation is a very simple method and can be employed for routine analysis of these two drugs in combined dosage form using simple instrumentation. Once the molar absorptions are determined, very little time will be required for routine analysis, as it would only require
5.
4. Gen, S. H. and Ismail, R., Validation of HPLC method for tramadol and O-desmentyl tramadol in human plasma using solid phases extraction, J. Chromatogr. B., 759 (2), 325 (2001). Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, Vol. II, 1996, Published by the Controller of Publication, New Delhi, (1996), p. 484, 554. British Pharmacopoeia, 1993, Vol. I, International Edition, Published on the recommendation of the Medicines Commissions Pursuant to the Medicines Act, (1968), p. 429, 483. USP 24, NF 19, U. S. Pharmacopoeia and National Formulary, Asian edition, U. S. Pharmacopoeial convention, INC, (2000) p. 17-18, 2152.
6.
7.
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First Order Derivative Spectrophotometric Determination of Nebivolol in Bulk and Tablets

Atul A. Shirkhedkar*, Prasad M. Bugdane, Sanjay J. Surana
Abstract: A simple rapid, accurate and economical First order UV-derivative spectrophotometric method has been developed for estimation of Nebivolol from pharmaceutical formulations. In methanol-water (3:7) solvent, Nebivolol showed maximum absorbance at 281 nm. In first order derivative spectrum amplitude of the trough was recorded at 291nm. Linearity was observed at 291 nm in the concentration range of 10 - 70 g/mL (r2=0.9997). The amount of drug estimated from the tablet formulation was found to be in the good agreement with the label claim. The method has been validated statistically and by recovery studies.
Introduction
Nebivolol hydrochloride (NEB), 1-(6-flurochroman-2yl) {[2-(6flurochroman-2-yl)-2 hydroxyethyl] amino} ethanol, Hcl is antihypertensive.1 Literature survey revealed few analytical methods which include liquid chromatography with tandem mass spectrometry,2 liquid chromatography coupled with electro spray ionization tandem mass sectroscoy3 for estimation of Nebivolol in biological fluids. RP-HPLC method has also been reported for estimation of Nebivolol from tablet formulations.4 Nebivolol is not official in IP, BP and USP. The aim of the present work is to develop First order spectrophotometric method and validate it for linearity, accuracy, precision, ruggedness and robustness.5
Preparation of sample solution For analysis of commercial formulation; twenty tablets were weighed, average weight determined and crushed into fine powder. A quantity of powder equivalent to 10 mg of Nebivolol was transferred into 50.0 mL volumetric flask containing 15 mL of methanol-water (3:7), shaken manually for 10 min., volume was adjusted to mark with same solvent and filtered through Whatmann filter aper no. 41 After appropriate dilutions, the amplitude of trough was recorded at 291nm. The concentration of the drug was calculated from linear regression equation. Recovery studies To study the accuracy and reproducibility of the proposed methods, recovery experiments were carried out by adding a known amount of drug to preanalysed sample at three levels and the percentage recoveries were calculated; the results are summarized in Table 1.

All the reagents were used of analytical grades. Preparation of standard stock solution Standard stock solution was prepared by dissolving 10 mg of NEB in 100mL of methanol-water (3:7) to get concentration of 100g/mL. Different aliquots were taken from the stock solution and diluted to 10 mL mark with same solvent to obtain series of concentrations. The solutions were scanned on spectrophotometer - 2450 (Shimadzu) in the UV range. The zero order spectrum showed maximum absorbance at 281 nm. The same spectrum was derivatised using UV probe software of instrument at Dl=2; the amplitude of trough was recorded at 291 nm. The calibration curve was found to be linear in the concentration range 10-7 g/mL (Y=0.00150 X -0.00034; r2 =0.9997).

The l max of NEB in methanol-water (3:7) was found to be 281 nm. The same spectrum was derivatised into first order; showed amplitude at 291 nm. The drug follows linearity in the concentration range of 10-70 g/mL (r2 =0.9997). The analysis of tablet formulation by proposed method was in good agreement with label claim. The method was validated for accuracy, precision, ruggedness and robustness. The recovery studies were carried out at three different levels i.e. 80%. 100% and 120%. The mean percentage recovery was found to be 100.15% (Brand-I) and 99.43% (Brand-II). The low values of %RSD are indicative of the accuracy and reproducibility of the method. The
R.C. Patel College of Pharmacy, Shirur, Dist: Dhule (M.S.). Author for correspondence: atul_shirkhedkar@yahoo.com
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precision of the method was studied as an intra-day, inter-day precision and repeatability. The % RSD value less than 2 indicate that the method is precise. Ruggedness of the proposed method was studied with the help of two analysts. The results from valudation studies are shown in Table 1.
Table 1: Validation of first order derivative spectrophotometric method Parameters Linearity and range (g/mL) Regression equation (Y= m X + C) LOD (g/mL) LOQ (g/mL) *Accuracy (%Recovery) Precision (%RSD) Inter-day Inter-day #Repeatability (%RSD) Ruggedness (%RSD) *mean of nine estimation; #mean of six estimations 1.33 1.44 1.65 1.80
3. 4. 5.
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Conclusion
The proosed method is simple, rapid, accurate, economical and useful for the routine analysis of Nebivolol from marketed formulation.
Acknowledgement
The authors are thankful to R.C. Patel College of Pharmacy; Shirpur, for providing facilities to carry out this work.
Results 10-70 0.00150 X - 0.00034 2.74 8.30 99.43-100.15
Reference
1. 2. The Merck Index, 12th Edn., Merck & Co., Inc., Whitehouse station, 1994, 1103. Thevis M., Opfermann G., Schanzer W., Biomed. Chromatogr., 2001, 15,393-402. Ramakrishna N.V.S., J. of Pharma.And Bio. Ana., 2005, 39 (5,4), 10061013. Sankar G.G. et al., Asian J. of Chem., 2005, 17 (2), 1259-1263. United States Pharmacopeia, 28th edn, United State Pharmacopoeial Convention, Inc., Rockville, 2005, 2749
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Application of Oxidants to the Spectrophotometric Determination of Gemifloxacin Mesylate in Pharmaceutical Formulations

Marothu Vamsi Krishna*, Dannana Gowri Sankar
Abstract: Four new spectrophotometric methods for the determination of gemifloxacin mesylate (GFX) have been proposed. The first three methods. i.e. A, B and C, are based on the oxidation of the drug with Fe (III) and the estimation of Fe (II) produced after chelation with either 1,10-phenanthroline or 2,2'-bipyridyl or ferricyanide at 515, 520 and 760 nm, respectively. The beer's law was obeyed in the concentration ranges of 3-15, 4-20 and 2-10 g ml-1 with molar absorptivity of 3.55 x 104, 2.10 x 104 and 3.10 x 104 L mole-1 cm-1for methods A, B and C respectively. The fourth method, i.e. D was based on the interaction of GFX with ammonium heptamolybdate tetra hydrate, which resulted in the formation of molybdenum blue with lmax 825 nm. The linear dynamic range and the molar absorptivity values were found to be 6-30 g ml-1 and 1.38 x 103 L mole-1 cm-1, respectively. The results of the proposed methods were validated statistically and are found to be accurate and precise. The proposed methods were applied successfully to the determination of GFX in commercial tablets.
Introduction
Gemifloxacin, (R,S)-7-(3-aminomethyl-4-syn-methoxyimino-1pyrrolidinyl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-1,8naphthyridine-3-carboxylic acid methanesulfonate, is a new fluoroquinolone antibacterial compound with enhanced affinity for bacterial topoisomerase IV and is being developed for the treatment of respiratory and urinary tract infections. The compound has a broad spectrum of activity against Gram-positive and Gram-negative bacteria.1-3 Literature survey revealed that few analytical methods have been reported for the estimation of GFX; they include high-performance liquid chromatographytandem mass spectrometry (LC-MS-MS), 4-5 microchip electrophoresis,6 chiral high-performance liquid chromatography7 and chiral counter-current chromatography.8 To the best of our knowledge, there is no work in the literature reported about the spectrophotometric method for the analysis of GFX in either biological fluids or pharmaceutical formulations. Hence the author has made an attempt to develop four simple and rapid spectrophotometric methods for the estimation of GFX in bulk drugs and in pharmaceutical formulations. The first three methods. i.e. A, B and C, are based on the oxidation of the drug with Fe(III) and the estimation of Fe (II) produced after chelation
with either 1,10-phenanthroline or 2,2'-bipyridyl or ferricyanide at 515, 520 and 760 nm, respectively. In the fourth method, molybdate is reduced to molybdenum blue which is determined at 825 nm.
Experimental
Apparatus: All spectral and absorbance measurements were made on a systronic model 117 digital spectrophotometer with 10mm matched quartz cells. Materials and reagents: All chemicals used were of analytical reagent grade and double distilled water was used for preparing the reagent solutions. GFX was obtained from Hetero drugs, Hyderabad. Stock solution of GFX was freshly prepared by dissolving 100mg of GFX in 100ml of distilled water and then this was further diluted with distilled water so as to obtain working standard solution of 100 g/ml for methods A, B and C, 300 g/ml for method D.1, 10-phenanthroline (0.2%) was prepared by dissolving 200 mg of 1, 10-phenanthroline in 100 ml of distilled water with warming. Ferric chloride (0.2%) was freshly prepared by dissolving 200 mg of ferric chloride in 100 ml of distilled water. Orthophosphoric acid solution was prepared by mixing 8.5 ml of orthophosphoric acid with distilled water and final volume make
Pharmaceutical Analysis and Quality Assurance Division, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam. Author for correspondence E-mail: marothu_vamsi@rediffmail.com
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up to 1000 ml. 2, 2'- bipyridyl (0.5%) was prepared by dissolving 500 mg of 2, 2'- bipyridyl in 100 ml of distilled water. Potassium ferricyanide (0.1%) was prepared by dissolving 100 mg of potassium ferricyanide in 100 ml of distilled water. 2.5 x 10-2 M ammonium hepta molybdate tetra hydrate solution was prepared in 4 M sulphuric acid. Disodium hydrogen phosphate- Citric acid buffer solution of varying pH values were prepared by mixing appropriate volume of 0.2 M disodium hydrogen phosphate and 0.1 M citric acid. The pH was adjusted with the aid of pH meter.
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General Procedure and Calibration

Method A: Different aliquots of working standard solution (100 g ml-1) from 0.3-1.5 ml were transferred in to a series of 10 ml standard flasks. To each flask 1.5 ml of ferric chloride and 1.5 ml 1,10-phenanthroline were added and kept in a water bath (60 10C) for 15 min, then immediately cooled to room temperature (2510 C) using a cold water bath and 1 ml of o-phosphoric acid was added. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 510 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation. Method B: Different aliquots of working standard solution (100 g ml-1) from 0.4-2.0 ml were transferred in to a series of 10 ml standard flasks. To each flask 1.5 ml of ferric chloride and 2.0 ml 2,2'-bipyridyl were added and kept in a water bath (60 10 C) for 15 min, then immediately cooled to room temperature (25 10 C) using a cold water bath and 1 ml of o-phosphoric acid was added. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 520 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation. Method C: Different aliquots of working standard solution (100 g ml-1) from 0.2-1.0 ml were transferred in to a series of 10 ml standard flasks. To each flask 1.5 ml of ferric chloride and 1.0 ml potassium ferricyanide were added, mixed well and let stand for 10 min. Finally 1 ml of 1N HCl was added to each flask and made up to volume with distilled water and mixed well. The absorbance of each solution was measured at 760 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.
Method D: Different aliquots of working standard solution (300 g ml-1) from 0.2-1.0 ml were transferred in to a series of 10 ml standard flasks. To each flask 2.5 ml of ammonium hepta molybdate tetra hydrate and 5.0 ml of disodium hydrogen phosphate- citric acid buffer solution of pH 4.0 were added and kept in a water bath (6010 C) for 15 min, then immediately cooled to room temperature (2510 C) using a cold water bath. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 825 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation. Procedure for Tablets: Twenty tablets were weighed accurately and ground in to a fine powder. An amount of powder equivalent to 30 mg of GFX was weighed into a 100 ml volumetric flask, about 40 ml of water containing 10 ml of the methanol were added and shaken thoroughly for about 20 min, then the volume was made up to the mark with the water , mixed well and filtered using a quantitative filter paper. The assay of the tablets was completed according to the general procedure.

The optical characteristics such as Beer's law limits, Sandell's sensitivity, molar absorptivity, percent relative standard deviation (calculated from eight replicate samples containing th of the amount of the upper beer's law limits) were calculated for all the methods and the results are summarized in table 1. Regression characteristics like standard deviation of slope (Sb ), standard deviation of intercept (Sa ), standard error of estimation (Se ), % range of error (0.05 and 0.01 confidence limits) and detection limit were calculated for all the methods and are shown in table 1. Commercial formulation of GFX was successfully analyzed by the proposed methods. The reliability of the proposed method was checked by standard addition method. The results (Table 2) show that the mean recoveries were 99.90% by all the methods.Interference studies revealed that the common excipients and other additives usually present in dosage form did not interfere in the proposed methods. The performance order of the proposed methods is Method C> Method A > Method B > Method D. In conclusion the proposed spectrophotometric methods for the estimation of GFX can be used for the routine quality control of the drug in bulk as well as in pharmaceutical formulations.
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Table 1: Optical, Regression Characteristics of the Proposed Methods for GFX
Parameter gmax (nm) Method A 510 Method B 520 Method C 760 Method D 825
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References
1.
Oh J I, Pack M J, Ahn M Y, Kim CY, Hong C Y, Kim I C and Kwak J H (1996) In vitro and in vivo evaluations of LB Beer's law limits (g ml-1) 3.0 15.0 4.0 20.0 2.0 - 10.0 6.0 - 30.0 20304, a new fluoronaphthyridone. Antimicrob Agents -1 Chemother. 40; 1564-1568. Detection limits (g ml ) 0.102 0.097 0.053 0.216 2. Cormican M G and Jones R N (1997) Antimicrobial activity Molar absorptivity (L mole-1 cm-1) 2.70 x 104 2.06 x 104 4.27 x 104 1.38 x 104 and spectrum of LB 20304, a new fluoronaphthyridone. Sandell's sensitivity 0.017 0.0023 0.011 0.035 Antimicrob Agents Chemother. 41; 204-211. (g cm-2 / 0.001 absorbance unit) 3. Hohl A F, Frei R, Ponter V, Von graevenitz A, Knapp C, Washington J, Johnson D and Jones R N (1998) Regression equation (Y = a + bC) -2 -2 -2 -2 International multicenter investigation of LB 20304, a new Slope (b) 5.5 x 10 4.2 x 10 8.7 x 10 2.8 x 10 fluoronaphthyridone. Clin Microbiol Infect. 4; 280-284. -3 -3 -3 -3 Standard deviation of slope (Sb) 0.19 x 10 0.10 x 10 0.24 x 10 0.10 x 10 4. Doyle E, Fowles S E, Mc Donnell D F, Mc Carthy and White S A (2000) Rapid determination of gemifloxacin in Intercept (a) 1.3 x 10-3 0.40 x 10-3 -0.50 x 10-3 -0.40 x 10-3 human plasma by high-performance liquid -3 -3 -3 -3 Standard deviation of intercept (Sa) 1.91 x 10 1.38 x 10 1.57 x 10 2.06 x 10 chromatography-tandem mass spectrometry. Journal of chromatography B. 746; 191-198. Standard error of estimation (Se) 1.82 x 10-3 1.32 x 10-3 1.49 x 10-3 1.97 x 10-3 5. Ramji J V, Austin N E, Boyle G W, Chalker M H, Duncan G, Correlation coefficient (r) 0.9999 0.9999 0.9999 0.9999 Fairless A J, Hollis F J, Mc Donnell D F, Musick T J and a Relative standard deviation (%) 0.077 0.109 0.107 0.108 Shardlow P C(2001) The disposition of gemifloxacin,a new fluoroquinolone antibiotic in rats and dogs. Drug % Range of error(Confidence limits)a Metabolisam and Disposition. 29; 435-442. 0.05 level 0.065 0.066 0.090 0.091 6. Seung I l Cho, Jiyeon Shim, Min-su kim, Yong-kweon kim, 0.01 level 0.096 0.097 0.133 0.134 Doo soo chung (2004) On-line sample clean up and chiral separation of gemifloxacin in a urinary solution using % Error in bulk samplesb 0.056 0.073 0.107 -0.055 chiral crown ether as a chiral selector in microchip a Average of eight determinations. electrophoresis. Journal of chromatography A. 1055; b Average of three determinations 241-245. In Y =a + bC, Y is absorbance and C is concentration 7. Won jae Lee, Chang Yang Hong (2000) Direct liquid chromatographic enantiomer Table 2: Results of recovery study by standard addition method separation of new fluoroquinolones Dosage Labeled % Recovery* % RSD including gemifloxacxin. Journal of -1 forms (g ml ) chromatography A. 879; 113-120. Taken+ 8. Eun sook kim, yoo-mo koo, doo soo chung. Added Chiral counter-current chromatography of Method A Method B Method C Method D Method A Method B Method C Method D gemifloxacin guided by capillary electrophoresis using (+)- (18-crown-6)Tablets-1 4+4 100.18 99.90 99.98 100.01 0.35 0.24 0.28 0.52 tetracarboxylic acid as a chiral selector. 5+5 100.10 100.32 100.60 100.20 0.41 0.21 0.53 0.36 Journal of chromatography A. 1045; 119Tablets-2 3+5 99.99 100.05 100.10 100.02 0.26 0.52 0.58 0.40 124. 4+6 100.04 100.16 100.25 100.32 0.48 0.31 0.45 0.64 Source: The Pharma Review, * Average of six independent analyses
December 2007
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Simultaneous Estimation of Aceclofenac and Paracetamol in Combined Dosage Form by Two Wavelength Spectrophotometry
Wadher S. J.*, Momin M. Y., Puranik M. P. and Yeole P. G.
Abstract: A simple, rapid and economical method for simultaneous estimation of aceclofenac and paracetamol in tablet formulation has been developed. Paracetamol is quantized using two wavelengths, 230 nm and 275 nm where as aceclofenac is quantized using two wavelengths, 224 nm and 269 nm in methanol. The Beer's law is obeyed by paracetamol and aceclofenac in concentration range of 0-30 g/ml and 0-50g/ml for aceclofenac and paracetamol respectively.
Introduction
Aceclofenac (ACF), {[2-(2`, 6`-dichlorophenyl) amino] phenyl acetoxyacetic acid} is a new phenyl acetic acid derivative with potent analgesic and anti-inflammatory properties with improved gastric tolerance. Paracetamol (PCM), chemically 4-hydroxy acetanilide, is a centrally and peripherally acting analgesic, antipyretic agent. A combination of these two drugs containing aceclofenac (100 mg) and paracetamol (500 mg) is available commercially by different manufacturers. This combination is use for pain and management of rheumatic disorders. B.P.1 describes titrimetric method for estimation of aceclofenac. Paracetamol is official in I.P.2, B.P.1 and U.S.P.3 which describes titrimetric and spectrophotometric methods for its estimation. Only few methods4-9 have been reported for determination of aceclofenac individually, whereas many methods9-13 have been described in literature for determination of paracetamol alone or in combination with other drugs. The review of the literature revealed that no method is as yet reported for the simultaneous estimation of both the drugs in combined dosage form. Present paper described simple, accurate and reproducible two wavelength spectrophotometric method for simultaneous estimation of two drugs in tablet formulation.
used to measure absorbance of the resulting solution, A Shimadzu analytical balance model AW 220. Reagent Methanol AR grade.
Standard stock solution

1. Aceclofenac standard stock solution (0.1 mg/ml) was prepared by transferring accurately weighted 10 mg of pure aceclofenac in 100 ml volumetric flask and volume was made to mark with methanol to give 10 g/ml. 2. Paracetamol standard stock solution (0.5 mg/ml) was prepared by transferring accurately weighted 50 mg of pure paracetamol in 100 ml volumetric flask and volume was made to mark with methanol to give 50 g/ml. Selection of wavelength: Selection of analytical wavelengths was done by taking pure samples of aceclofenac and paracetamol which were separately dissolved in methanol to give 20g/ml of each drug. They were scanned in the wavelength range of 200 to 400 nm. From the overlain spectra (fig.1), the wavelengths selected for paracetamol were 230 nm and 275 nm where aceclofenac has same absorbance and for aceclofenac wavelengths selected were 224 nm and 269 nm, were paracetamol has same absorbance.

Instruments: A Shimadzu model 2401 double beam UV-visible spectrophotometer with a pair of 10 mm matched quartz cells was
Linearity
A series of different concentration in range of 0-50 g/ml for paracetamol and 0-30 g/ml for aceclofenac were prepared from
Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha, M.S. *Author for correspondence Email: sjwadher@yahoo.com
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2.500
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Table 1: Summary of Validation Parameters Sr. No. Parameters PCM 1 Linearity Range (g/ml) Accuracy (%) Precision (R. S. D.) Ruggedness Interday (n=3) 99.12 99.89 0-30 g/ml 99.42 0.221 2 3 4
ACF 0-50 g/ml 99.52 0.235 99.70 99.50
2.000
A b s
1.500
1.000
Intraday(n=3)
0.500
Table 2: Simultaneous Assay of Aceclofenac and Paracetamol in Pharmaceutical Dosage Form Sr. No. Aceclofenac Paracetamol
250.0 300.0 Wavelength (nm.) 350.0 400.0
200.0
Claim (mg) 1. 2. 100 100
Found (mg)* 99.52 100.10
Recovery (%) ** 99.88 99.71
Claim (mg) 500 500
Found Recovery (mg)* 497.90 499.20 (%)** 100.02 99.78
Fig. 1: Overlain spectra of paracetamol and aceclofenac.
stock solution. The absorbances of aceclofenac were noted at 224 nm and 269 nm and the absorbance of paracetamol were noted at 230 nm and 275 nm. The difference in absorbance of aceclofenac and paracetamol at these two wavelengths was calculated and calibration curve were plotted as absorbance difference verses concentration for aceclofenac and paracetamol. The curve shows linearity in the range of 0-35 g/ ml and 0-50g/ml for aceclofenac and paracetamol respectively.
* Average of three determination. ** After spiking the pre-analyzed sample at different levels.
Sample solution
Twenty tablets were weighed accurately, the average weight determined and then ground to a fine powder. A quantity equivalent to 100 mg of paracetamol and 20 mg of aceclofenac was weighed and transferred to a 100 ml volumetric flask. The content was shaken with methanol to dissolve the active ingredients, made to volume and filtered through Whatman filter paper no 41. The filtrate was diluted with methanol to achieve final concentration of 50 g/ml for paracetamol and 10g/ml of aceclofenac.
paracetamol in tablet formulation. The standard deviation values were satisfactory low and recovery was closed to 100 % indicating the reproducibility, accuracy and precision of proposed method. The linearity performed for both the drugs and the absorbencies were found to be linear in concentration range of 0-35 g/ ml and 050g/ml for aceclofenac and paracetamol respectively.
Conclusion
The results indicates that the proposed two wavelength method was simple, accurate and precise, hence it can be use for routine analysis which would involve determination of absorbance of sample and standard solution at the four wavelengths and simple calculation.
References
1. British Pharmacopoeia, Her Majesty's Stationary office, London, UK, 2000, 32-33. 2. Indian Pharmacopoeia, Government of India, Ministry of Health and Family welfare, Vol. II, Published by the controller of publication Delhi, 1996, 484, 554-555. 3. USP 24, NF19, The United States Pharmacopoeia & National formulary, XXIV, U.S. Pharmacopoeia convention, Inc., Rockville, 2000, 17-18. 4. Hasan, N.Y., Abdel Elkawy, M. and Wagieh, N.E., Stability indicating methods for the determination of Aceclofenac, Farmaco., 2003, 58, 9199. 5. Zawilla, N.H. and Mohammad, M.A., Determination of in bulk and pharmaceutical formulations, J. Pharm. Biomed. Anal., 2002, 27, 243251. 6. Hinz, B., Auge D., Rietbrock, S. and Weren, U., Simultaneous determination of Aceclofenac in human plasma by high performance liquid chromatography, J. Biomed. Chromatogr., 2002, 17, 268-275.
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Results
The results obtained by proposed method were in good agreement with the label claim (Table 1). The additives and excipient did not in interference in analysis of the label by proposed method. The method was validated in terms of accuracy, precision, ruggedness, linearity and range. The accuracy of the proposed method was determined by recovery studies. The summery of validation parameter is shown in Table 2.
Discussion
The present study was carried out to develop a simple, rapid, sensitive, accurate, precise and rugged spectrophotometric method for the simultaneous estimation of aceclofenac and
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7. Sharty, Y.S., Refaat, M. and Khateeb S.Z., Stability indicating spectrophotometric and densitometric methods for determination of Aceclofenac, Drug Dev. Ind. Pharm., 2002, 28, 571 582. 8. Lee, H. S., Jeong, C. K., Choi, S. J., Kim, S. B. and Lee M. H., Simultaneous determination of Aceclofenac and Diclofenac in human plasma by narrow bore HPLC using column switching, J. Pharm Biomed. Anal., 2000, 23, 775 781. 9. Harish, L., Rau, A. R. and Arora, P., Simultaneous estimation of Paracetamol and Diclofenac sodium by HPLC, Indian Drugs, 1991, 28, 285-286. 10. Subramanian, G., Musmade, P. and Udupa, N., Simultaneous estimation of Tizanidine, Diclofenac potassium and Paracetamol in tablet, Ind. J. Pharm. Sci., 2004, 66, 694-693. 11. Patil, D., Raman, B., Simultaneous estimation of Dextropropoxyphen HCl, Diclofenac sodium & Paracetamol by RP-HPLC, Indian Drugs, 2001, 38(1), 36-39. 12. Marin, A., Garcia, A. and Barbas, C., Validation of a HPLC quantification of Acetaminophen Phenylephrine and Chlorpheniramine in pharmaceutical formulations capsules and sachets, J. Pharm, Biomed. Anal., 2002, 29, 701-714. 13. Garcia, A., Ruperez, F. J., Marin, A. and Barbas, C., Poly (ethyleneglycol) column for the determination of Acetaminophen, Phenylephrine and Chlorpheniramine in pharmaceutical formulations, J. Chromatogr., B., 2003, 785, 237-293. 14. ICH Q2A, International Conference of Harmonization: Guidelines for Industry Q2A, Text on the Validation of Analytical Procedures, Federal Register, 1996, 60, 11260-11262.
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Application of UV-Spectrophotometric Method for Estimation of Drotaverine Hydrochloride in Bulk & Tablets
A. A. Shirkhedkar*, G. H. Upasani and S.J. Surana
Abstract: A simple, rapid and accurate spectrophotometric method has been developed for the quantitative estimation of Drotaverine HCl (DH) in bulk and tablets. In methanol, DH exhibits absorption at 354 nm and the method obeys Beer's law at the concentration range of 4 - 24 g/mL. The percentage label claim was found to be in range of 98-101%. The proposed method was validated statistically and by recovery studies.
Introduction
Drotaverine hydrochloride (DH), 1- (3, 4-diethoxybenzylidene)-6, 7 diethoxy 1, 2, 3, 4- tetrahydroisoquinoline is antispasmodic1 and acts by inhibiting phosphodiesterase IV enzyme, specific for smooth muscle spasm and pain, mainly in labor pain.2-3 Literature survey revealed that HPLC method for the determination of DH in human plasma and urine4 and few spectrophotometric methods5-6 are available for estimation of DH alone and in combinations with other drugs. DH is not official in pharmacopoeia. Therefore, it was thought worthwhile to develop a simple, accurate and reliable spectrophotometric method for the estimation of DH in bulk and in tablet dosage form using methanol as a solvent. All the chemicals used were of analytical grade. Spectral and absorbance measurements were made on Shimadzu UV- visible spectrophotometer- 2450 with 10mm matched quartz cells.
Table1: Optical Characteristics and Precision of The Proposed Method
Parameters Wavelength Beer's law limit (g/ml) Regression equation (Y= mx+C) Molar extinction coefficient (Lmol cm )* E 1% Slope (m) Intercept (c) Correlation coefficient (r)
-1/ -1
354 nm 4 - 24 Y=0.02516x+0.0115 1.06104 230 0.0251 0.0115 0.9994
Average of six determinations*
Preparation of standard solution

Accurately weighed 10 mg of DH pure drug was dissolved in methanol to give stock solution of 100 g/mL concentration. From this stock solution, working standard solutions of drug (4-24 g/mL) were prepared by appropriate dilutions with distilled water. Working standard solutions were scanned in the UV range of (200-400nm). The optical characteristics such as Beer's law limits, molar extinction coefficient (Lmol-1/cm-1), regression equation, correlation coefficient were calculated and results are presented in Table 1.
powder. An accurately weighed powder equivalent to 10 mg of DH was transferred in to 100 mL volumetric flask and volume make up to the mark with methanol. From this stock solution, working sample solutions of drug were prepared by appropriate dilutions with distilled water. The results of assay are presented in Table 2. Table 2: Results of Assay
Label claim mg/tab 40 *Amount found mg/tab 39.9 %Label claim 99.7 % R.S.D. 1.27
Average of three determinations*
Recovery studies
Accuracy of the method was checked by recovery studies. The recovery studies were carried at 80%, 100% and 120% level. The results of recovery studies are presented in Table 3.
Preparation of sample solution

Twenty tablets were accurately weighed and ground to fine

In methanol, DH exhibits absorption at 354 nm. The linearity was observed in concentration range of 4-24 g/mL. The amount of
R.C. Patel College of Pharmacy, Shirpur Dist: Dhule (M.S.) *Author for Correspondence E-mail: atul_shirkhedkar@yahoo.com
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Table 3: Results of Recovery Studies
Concentration Added (mcg/mL) 8 9.9 101 *Amount recovered (mcg/mL) 10 12.1 1.3 % Recovered % R.S.D 7.9 99.8 1.7
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12 99.6 1.5
Average of three determinations at each level*
DH estimated by the proposed method was in good agreement with the label claim. The % recovery was found to be in the range of 99-101%. The low % RSD value indicates that method is accurate. The proposed method is simple, accurate, economical and be used for routine analysis of DH from tablet formulations.
References
1. 2. 3. 4. 5. 6. Martindale, The Complete Drug Reference, 33rd edn. Sean C. Sweetman, The Pharmaceutical Press, London, 1606. The Merck Index, 13th edn., Merck & Co., Inc., Whitehouse station, 2004, 609-610. Singh K.C., Jain P., Goel N. and Saxena A., Inter. J. Gayneco. & Obst. 2004, 84 (1), 17-21. Bolaji O.O., Onyeji C.O., J.Chromatogr.B. Biomed. App., 1993, 622 (1), 93-97. Mahajan V.K., Dahivelkar P.P., Fursule R.A., Shirkhedkar A.A and Surana S.J., Indian Drug, 2006, 43 (8), 656-659. Dahivelkar P.P., Mahajan V.K., Bari S.B., Shirkhedkar A.A., and Surana S.J., Indian Drugs, 2006, 43 (11), 896-900.
Source: The Pharma Review, August 2007
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Oxidative Coupling, Complex Formation and Internal Salt Formation Reactions for Visible Spectrophotometric Determination of Alfuzosin Hydrochloride in Pharmaceutical Formulations
M. Vamsi Krishna*, D. Gowri Sankar
Abstract: Three simple and sensitive visible spectrophotometric methods have been developed for the determination of Alfuzosin hydrochloride (AFZ) in pharmaceutical formulations. Method A is based on the oxidative coupling reaction of AFZ with thymol under alkaline conditions in presence of sodium hypochlorite and the colored product formed was measured at 510 nm. Method B is based on the reaction of AFZ with cobalt thocyanate and the colored complex was measured at 630 nm. Method C is based on the formation of a colored internal salt between AFZ and citric acid in presence of acetic anhydride, which absorbs maximally at 580 nm. Beer's law is obeyed in the concentration ranges 8-40, 20-100 and 5-25 g/ml for method A, B and C respectively. The methods have been applied to the determination of AFZ in commercial tablets. Results of analysis were validated statistically and are found to accurate and precise.
Introduction
Alfuzosin Hydrochloride (AFZ) 1 is a alpha 1- receptor blocker and is chemically known as N-[3-[(4-amino-6, 7-dimethoxy quinazolin-2-yl)-methyl-amino]propyl]oxolane-2-carboxamide hydrochloride. Figure 1 shows the structure of alfuzosin. It is used for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia. Literature survey reveals that, few chromatographic2-6 methods have been reported for the estimation of AFZ. To the best of our knowledge, there is no work in the literature reported about the spectrophotometric method for the analysis of AFZ in either biological fluids or pharmaceutical formulations. Hence the author has made an attempt to develop three simple and sensitive spectrophotometric methods for the
NH2
estimation of AFZ in bulk drugs and in pharmaceutical formulations. Method A is based on the oxidative coupling reaction of AFZ with thymol under alkaline conditions in presence of sodium hypochlorite and the colored product formed was measured at 510 nm. Method B is based on the reaction of AFZ with cobalt thocyanate and the colored complex was measured at 630 nm. Method C is based on the formation of a colored internal salt between AFZ and citric acid in presence of acetic anhydride, which absorbs maximally at 580 nm.
Experimental
Apparatus: All spectral and absorbance measurements were made on a systronic model 117 digital spectrophotometer with 10mm matched quartz cells. Materials and reagents: All chemicals used were of analytical reagent grade and double distilled water was used for preparing the reagent solutions. AFZ was obtained from Dr. Reddy's labs Hyderabad. Stock solution of AFZ was freshly prepared by dissolving 100mg of AFZ in 100ml of distilled water and then this was further diluted with distilled water so as to obtain working standard solution of 200, 400 and 250g/ml for methods A, B and C
CH3O
N O O
CH O 3
NH
CH 3
Figure 1: Structure of Alfuzosin
Pharmaceutical Analysis and Quality Assurance Division, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam. *Author for Correspondence: E-mail: marothu_vamsi@rediffmail.com
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respectively. H2SO4 (50%), Aqueous NaOCl, 5% Thymol (5 g in 100 ml of ethanol), NaOH solution (40%), Cobalt thiocyanate solution (2.50x10 -1M), Buffer solution (pH 2.0), nitrobenzene and 12% citric acid in acetic anhydride were used.
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General procedure
Method A: Aliquots of working standard solution (0.4-2.0 ml, 200 g/ml) were placed separately in a series of 10 ml graduated test tubes. To that 0.4 ml of H2SO4 , 1.0 ml sodium hypochlorite solution and 0.5 ml of thymol solution were added and kept a side for 5 min at room temperature with occasional shaking. Then 1.2 ml of NaOH solution was added and all the tubes were put in water bath at 20-25 0C for 5 min and made up to 10 ml with distilled water. The absorbances were measured at 510 nm against the reagent blank. The calibration curve was constructed by plotting the absorbance versus final concentration of the alfuzosin. The content of the unknown was computed either from calibration curve or regression equation.
Assay of pharmaceutical tablets: Twenty tablets were powdered and mixed thoroughly. An amount equivalent to 100 mg of the drug was dissolved in water and filtered. The filtrate was made up to 100 ml and appropriate aliquots of the drug solution were treated as described above for the determination of AFZ.

Analytical data: The optical characteristics such as Beer's law limits, Sandell's sensitivity, molar absorptivity, percent relative standard deviation (calculated from eight replicate samples containing 3/4th of the amount of the upper beer's law limits) were calculated for all the methods and the results are summarized in table 1. Regression characteristics like standard deviation of slope (Sb ), standard deviation of intercept (Sa ), standard error of estimation (Se ), % range of error (0.05 and 0.01 confidence limits) and detection limit were calculated for all the methods and are shown in table 1.
Method B: Aliquots of working standard solution (0.5-2.5 ml, Table 1. Optical and Regression Characteristics, Precision and Accuracy of the 400 g/ml) were delivered in to a series of calibrated tubes. 2 Proposed Methods for AFZ Parameter Method A Method B Method C ml of buffer of pH 2.0 and 5 ml of cobalt thocyanate solutions gmax (nm) 510 630 580 were added and the total volume in each tube was adjusted to 15 ml with distilled water. These solutions in the tubes were Beer's law limits (g ml-1) 8.0 - 40.0 20.0 - 100.0 5.0 - 25 transferred to 125 ml separating funnel. To each separating -1 Detection limits (g ml ) 0.190 0.771 0.143 funnel 10.0 ml of nitrobenzene was added and the contents -1 -1 3 3 Molar absorptivity (L mole cm ) 9.1 x 10 3.83 x 10 1.50 x 104 were shaken for 2 min. The two phases were allowed to Sandell's sensitivity 0.046 0.110 0.028 separate and the absorbance of the separated nitrobenzene (g cm-2 / 0.001 absorbance unit) layer was measured after 20 min at 630 nm against the Regression equation (Y = a + bC) reagent blank. The calibration curve was constructed by Slope (b) 2.15 x 10-2 9.06 x 10-3 3.56 x 10-2 plotting the absorbance versus final concentration of the Standard deviation of slope (Sb) 0.05 x 10-3 0.04 x 10-3 0.10x 10-3 alfuzosin. The content of the unknown was computed either Intercept (a) -0.70 x 10-3 1.0 x 10-3 -1.70 x 10-3 from calibration curve or regression equation.
a Method C: Aliquots of working standard solution (0.5-2.5 ml, Standard error of estimation (Se) 1.82 x 10-3 2.22 x 10-3 250g/ml) were transferred in to a series of 25 ml graduated tubes and gently evaporated on a boiling water bath to Correlation coefficient (r) 0.9999 0.9999 a dryness. 10 ml of citric acid reagent was added to each tube. Relative standard deviation (%) 0.121 0.142 the tubes were placed in a boiling water bath and heated for a % Range of error(Confidence limits) 30 min. the solution in each tube was made up to the mark 0.05 level 0.102 0.120 with acetic anhydride, the absorbance of the colored 0.01 level 0.150 0.177 solutions was measured at 580 nm against the reagent blank. b % Error in bulk samples 0.164 -0.173 The calibration curve was constructed by plotting the a absorbance versus final concentration of the alfuzosin. The Average of eight determinations b content of the unknown was computed either from calibration Average of three determinations In Y =a + bC, Y is absorbance and C is concentration curve or regression equation.
Standard deviation of intercept (S ) 1.37 x 10-3 2.33 x 10-3
1.70 x 10-3 1.62 x 10-3 0.9999 0.130
0.109 0.161 0.211
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Analysis of pharmaceutical preparations: Application of the proposed methods to the determination of AFZ in its dosage forms was successfully made; the results are presented in table 2. The excellent recoveries obtained indicated the absence of any interference from the excipients.
Table 2. Results of analysis of tablet formulations containing AFZ Formulation Labeled Amount (mg) Tablets-1 Tablets-2 10 10 Recovery*%RSD** Method A 99.90.31 Method B Method C 4.
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References
1. Elhilali M M (2006) Alfuzosin: an alpha1-receptor blocker for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia. Expert. Opin. Pharmacother.7: 583-596. Niu C Q, Ren L M (2002) chiral separation and preparation of three new antagonists of alpha 1-adrenoceptors by chiral mobile phase HPLC. Yao Xue Xue Bao., 37: 450-453. Wiesner J L, Sutherland F C W, Van Essen G H, Hundt H K L, Swart K J, Hundt A F (2003) selective, sensitive and rapid liquid chromatography-tandem mass Spectrometry method for the determination of alfuzosin in human plasma. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 788: 361-368. Kratulovic A M, Vende J L (1989) Improved performance of the second generation alpha 1-AGP columns: applications to the routine assay of plasma levels of alfuzosin hydrochloride. Chirality. 1: 243-245. Rouchouse A, Manoha M, Durand A, Thenot J P (1990) Direct high performance liquid chromatographic determination of the enantiomers of alfuzosin in plasma on a second generation alpha 1-acid glycoprotein chiral stationary phase. J. Chromatogr., 506: 601-610. Guinebault P, Broquaire M, Colafranceschi C, Thenot JP (1986) High Performance liquid chromatographic determination of alfuzosin in biological fluids with fluorimetric detection and large volume injection. J. Chromatogr. 353: 361-369.
2.
3.
100.10.65 100.060.55
100.10.50 100.080.38 100.20.40
* Average of 5 determinations ** Relative standard deviation
Conclusions
The data given above reveal that the proposed methods are simple, accurate and sensitive with good precision and accuracy. The proposed methods can be used as alternative methods to the reported ones for the routine determination of alfuzosin. This encourages their successful use in routine analysis of alfuzosin in quality control laboratories
5.
6.
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Development of Spectrophotometric Analysis Method of Atenolol Gel Formulations

Nandy B. C, Gupta R.N
Abstract: Atenolol is a slightly water soluble drug which has about 50% bioavailability through the conventional oral route. It is the most commonly used selective b1 blocker in hypertension treatment. Generally, so many techniques are used to increase the aqueous solubilities of slightly water soluble drugs. To extract the drug candidates from the gel formulations organic solvents are used mostly as co-solvents. In this proposed method methanol used as a co-solvent with the phosphate buffer (pH 7.4) to extract the drug from the gel formulations. Atenolol shows maximum absorbance at near about 224 nm. Results of analysis were validated statistically and by the stability and recovery studies. The proposed investigation is new, simple, accurate, reproducible and also cost effective. Thus it can be successfully employed in routine analysis of atenolol gel formulations which can be applied for novel iontophoretic delivery system.
Introduction
Atenolol is a selective b1-adrenergic blocking agent used in the treatment of various cardiovascular disorders. With an oral bioavailability of 50%, the transdermal delivery of these drugs could be a potential alternative to oral delivery to increase therapeutic efficacy, bypassing hepatic first-pass metabolism and low oral absorption.1 Literature survey reveals determination of Atenolol by Spectrophotometric, HPLC, GC and Spectroflurimeteric methods. Although, most of the analytical methods of Atenolol formulations, they emphasized on HPLC estimation procedures. An attempt has been made in this proposed work to develop simple, cost effective and accurate UV and visible spectrophotometric methods for the estimation of drug from the gel formulations. The reported lmax for Atenolol drug in methanol at 224nm. So, many journals they used only methanol as a co-solvent to extract the drug contents from the various pharmaceutical formulations and also used simple spectrophotometric method, likes Norfloxacin,5 Clozapine,6 Citalopram Hydrobromide7 in tablets dosage form and Piroxicam,8 sodium nonivamide acetate II,9 b blockers10 & Atenolol11-13 in gel formulation.
2-4
Chemicals: The drug Atenolol was received as gift sample from Stadmed Pvt. Ltd., Kolkata and others reagents used all were analytical grades.
Scanning of Atenolol solution in various solvents

10mg of Atenolol was dissolved in 100ml of respective solvents phosphate buffer (pH7.4) and phosphate buffer (pH7.4) with 5% methanol solution), so as a solution of 100g/ml was prepared as a stock solution. From his 10ml was taken and the volume was made up to 50 ml to make solution concentration 20g/ml. The resulting solution was scanned by using spectrophotometer (Model Shimadzu, UV-Pharma spec 1700: UV-Visible Spectrophotophotometer). Scanning is reported in Fig. no. 1a and 2a respectively.
Preparation of standard plot of Atenolol in different solutions

10mg of Atenolol was dissolved in 100ml of different solution phosphate buffer (pH7.4) and phosphate buffer (pH7.4) with 5% methanol solution, so as a solution of 100g/ml was prepared as a stock solution. From his 2.5,5,7.5,10,12.5,&15ml was taken and the volume was made up to 50 ml to make solution concentration of 5,10,15,20,25,30g/ml respectively and absorbance were measured triplicate at wavelength maxima 224.2nm, 224.2 and 224.3 respectively. All standard curves are mentioned in Fig. no.1b and 2b in respective solutions.

Instrument: Shimadzu, UV-Pharma spec 1700: UV-Visible Spectrophotophotometer.
Department of Pharmaceutical Science, Birla Institute of Technology, Mesra, Ranchi

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Table 1: lmax values of atenolol solution in different solvents (concentration 20 g/ml)
S.No. Solvent 1 2
0.693 0.600
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l max in nm 224.2 224.3
(pH7.4) by heating on a water bath for 30 min to effect complete solution & keep it to cool at the room temperature & stirred that preparation using mixer until a clear gel were obtained. Methyl paraben and propyl paraben solution were prepared by heating with the some portion of propylene glycol until a clear solution were obtained. Then this solution was mixed with the gel preparation. Atenolol 1.5% w/w dispersed into the propylene glycol and l-menthol 2%w/w dissolved into the propylene glycol but in case of tween-20 incorporated to the polymer solutions directly and dissolved by continuous stirring at about 500 rpm for 30 min. the resulting solution was then adjusted to ph 7.4 by the addition of the 1M NaOH and/ or 1 M HCl solution. The solution was then stored at room temperature to ensure complete polymer dissolution in an air tight glass container.
Phosphate buffer ( pH7.4 ) Phosphate buffer ( pH7.4 ) with 5 % methanol

1.03340
3
Abs.
Abs.
2
0.400
0.50000
0.200
1
0.00000
0.000 210.000 220.000
240.000
260.000 nm.
280.000
300.000
0.00000
10.00000
20.00000 Conc. (mcg/ml)
30.00000
y=0.03113x+0.00566 r2 Correlation Coefficient = 0.99796
Fig. 1: (a) lmax values of atenolol of 20g/ml solution in Phosphate buffer solution (pH 7.4) was found at 224.2 and (b) its standard curve.
0.706 0.600 1.04772 1.00000
3
Abs.
0.400
Abs.
0.50000
Recovery studies
In order to check the accuracy, precision and the reproducibility of the proposed method, recovery studies were conducted.
0.200
2 1
0.000 210.000 220.000
240.000
260.000 nm.
280.000
300.000
0.00000 -0.097471 0.00000
10.00000
y=0.03113 x + 0.00965 r2 Correlation Coefficient = 0.99951
20.00000 Conc. (mcg/ml)
30.00000
Fig. 2: (a) lmax values of atenolol of 20g/ml solution in Phosphate buffer solution (pH 7.4) with 5% methanol was found at 224.3 and (b) its standard curve.
Solution Stability
To ensure the stability of the drug during the analysis as well as stability of drug in various formulations, a short term solution stability study was carried out.
The drug solution of strength 20g/ml and 30g/ml were prepared with the phosphate buffer (pH 7.4) and solutions pH were adjusted to 7.4 with the 1M HCL and/ or 1 M sodium hydroxide solutions as per required and the solutions were Table 2: Stability of the drug in phosphate buffer solution (pH7.4) kept at 37C for 48 hours and the samples withdrawn at 0, 24 Theoretical 20g/ml 30g/ml a n d 4 8 h o u r s . T h e s a m p l e s w e r e a n a l y z e d conc. of drug solution spectrophotometrically at 224.2nm
After accurately weighing 1 gm of gel preparation of different formulations (Table no.3), which contains equivalent to 15 mg of Atenolol was transferred to a 100ml volumetric flask. Now about 50 ml of respective solvent Phosphate buffer (pH 7.4) and another was phosphate buffer with 5% methanol were added separately and flask were shaken for about 15 min to solubilize the drug. Then the volumes were made up to the mark with respective solvents and which were used as stock solutions.
0 24 48 0 24 48 The tabulated results indicate that there was no significant Time interval (hrs) decrease in the absorbance after 48 hours. Hence the drug Absorbance at g 0.6267 0.6247 0.621 0.9407 0.9403 0.9383 was found to be stable in the media and also the % drug max 224.2 (n=3) 0.00125 0.00094 0.00081 0.00125 0.00125 0.00125 contents all are within the limit.
Procedure
% drug content
99.75 99.43 98.83 100.12 100.07 99.87 0.20 0.14 0.13 0.14 0.14 0.12 0.20 0.14 0.131 0.139 0.14 0.12
Sodium carboxy methyl cellulose based gel was prepared by % Coefficient Of dissolving polymer as required amount of phosphate buffer variation
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Preparation of gel formulations
Table 3: Formula used in different gel preparations of Atenolol.
S.N. Materials F1, F2 &F3 F4 ,F5 &F6 %(w/w) %(w/w) F7%(w/w) F8%(w/w) Blank Blank
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% Coeff. Of variation 0.253 0.130 0.252 0.100 0.253 0.137
Table 5: Recovery studies by using phosphate buffer solution (pH7.4) for Atenolol gel with l-menthol as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F4 F5 F6 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.)
1. 2. 3. 4. 5. 6. 7.
Atenolol Sodium CMC Methyl paraben Propyl paraben Propylene glycol L-Menthol Tween- 20
1.5 3.0 0.2 0.02 30 5
1.5 3.0 0.2 0.02 30 2 -
3.0 0.2 0.02 30 5
3.0 0.2 0.02 30 2 -
1ml up to 10ml 98.8690.253 2ml up to 10ml 99.6540.129 1ml up to 10ml 99.3690.251 2ml up to 10ml 99.6900.100 1ml up to 10ml 98.4400.250 2ml up to 10ml 99.5830.136
Then the solutions were filtered through Whatmann filter paper 41. The 1ml and 2ml filtrates of these each solutions were transferred to two different, 10 ml volumetric flasks separately and volume made up to 10 ml with the respective solvents. Blank preparations were also carried out by the same method of two different formulations containing respective penetration enhancers, which having no drug contents. Then the absorbance were noted at 224.2nm for phosphate buffer (pH 7.4) solution and 224.3 nm for phosphate buffer (pH 7.4) with 5% methanol solution, against corresponding blank preparation. Drug content and % recovery were calculated. (Table 4, 5,6 and 7).
Table 4: Recovery studies by using phosphate buffer solution (pH7.4) for Atenolol gel with tween-20 as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F1 F2 F3 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.) % Coeff. Of variation 0.100 0.015 0.174 0.087 0.031 0.100
Table 6: Recovery studies by using phosphate buffer solution (pH7.4) with 5 % methanol for Atenolol gel with tween-20 as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F1 F2 F3 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.) % Coeff. Of variation 0.333 0.263 1.050 0.260
1ml up to 10ml 100.360 0.170 0.169 2ml up to 10ml 100.1820.334 1ml up to 10ml 100.2980.264 2ml up to 10ml 100.860 1.06 1ml up to 10ml 99.870 0.260
2ml up to 10ml 101.200 0.190 0.187
Table 7: Recovery studies by using phosphate buffer solution (pH7.4) with 5 % methanol for Atenolol gel with l-menthol as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of %Recovery % formulations amount of Stock solution of Atenolol Coeff. Atenolol in (Mean S.D.) Of gel (mg) variation F4 F5 F6 15 15 15 15 15 15 1ml up to 10ml 99.7940.185 2ml up to 10ml 100.1820.061 1ml up to 10ml 100.2200.29 2ml up to 10ml 100.3200.26 1ml up to 10ml 99.5100.17 2ml up to 10ml 100.2200.22 0.185 0.060 0.289 0.259 0.170 0.219
1ml up to 10ml 99.3770.100 2ml up to 10ml 99.9700.015 1ml up to 10ml 99.4400.174 2ml up to 10ml 99.8320.087 1ml up to 10ml 99.8050.031 2ml up to 10ml 99.8650.100
Result and Discussion

The solubility of Atenolol in Phosphate buffer (pH 7.4) with 5% methanol solution was found to be higher as compared to its solubility in distilled water and phosphate buffer solution. It has also been seen that the solubility of Atenolol in distilled water and phosphate buffer is almost same. Thus it is concluded that the enhancement of solubility is due to the addition of the methanol which act as a co-solvent in the solution. Also it has been already
reported that Atenolol is freely soluble in methanol and slightly soluble in water. Hence, from the stability study of the atenolol in PB solution (pH 7.4), as per evident from the table 2), the % drug content after 48 hrs aged solution (kept at 37c temperature) of drug in PB (pH7.4) solutions were fount to have same and near by
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100% drug contains with out any degradation at pH 7.4 and also% coefficient of variation and standard deviation are very low. So the proposed method is accurate and which is statistically validated. This indicates that the analysis can be performed accurately with in 48 hrs of extraction of drug from the gel formulations. The results of recovery studies (table 4 and 5), which indicates that the % recovery by using PB (pH 7.4) gives the estimated, ranged from 99.377 0.10 to 99.865 0.10, in case of gel preparation where in tween-20 used as a penetration enhancer and for l-menthol 99.3690.251 to 99.869 0.2508. But the % recovery is almost 100% and above when used PB (pH7.4) with 5% methanol (table no. 6 and 7) as an extracting solvent, this indicates the accuracy of the proposed method. A value of standard deviation, % coefficient of variation are satisfactorily low and confirms further reproducibility, uniformity, consistent and precision of the proposed method. Although so many journals are used to determine the gel formulations via HPLC method, but widely used spectrophotometeric estimations of slightly water soluble drugs can be useful to perform in a precise manner. Although the g max values were used here at about 224.2nm and 224.3 nm, which are not affected by the water and methanol because the cut off wave lengths of that solvent are 180nm and 210 nm respectively. Since there are also other two peaks at around 275nm & 283nm, but these are having lower absorbance difference with increasing the concentration concurrently.
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References
1. Denet, A. R.; Ucakar, B.; Preat, V. Transdermal delivery of Timolol and Atenolol using electroporation and iontophoresis in combination: A mechanistic approach. Pharm. Res. 2003, 20, 1946-1951. Caplar, V.; Mihun, Z.M.; Hofman, H.; Kuftinee, J.; Kajfez, F.; Nagl, A.; Blazevic, N. Atenolol. In Analytical profiles of drugs substances. Florey, K. Ed. Acadmic press: New Yark, 1984; 13, pp 1-25. The Indian pharmacopoeia, Vol.1, Controller of publications: Delhi, 1996; pp 72-74. Dollery, C. Therapeutic Drugs. Ed. Churchill livirgstone: London, 1999; 1, PP A224-A227. Maheshwari R.K.Application of hydrotropic solubilization phenomenon in Spectrophotometeric estimation of Norfloxacin in tablets. Ind. J. Pharm. Edu. Res. 2006, 40, 237-240. Kuchekar B.S.; Gavhane Y. N.; Gaikwad N. B.; Thakkar S. V. Spectrophotometric estimation of Clozapine in tablets. Ind. J. Pharm. Edu. Res. 2006, 40,203-204. Pillai, S.; Singhvi I. Spectrophotometric methods for quantitative estimation of Citalopram Hydrobromide from tablet formulation. Ind. J. Pharm. Edu. Res. 2006, 40, 175-177. Attia , M. A., Gibaly, I. E., Shaltout, S.E., Fetih, G.N. 2004. Transbuccal permeation, anti-inflammatory activity and clinical efficacy of piroxicam formulated in different gels. Int. J. Pharm.276, 11-28. Fang, J.Y.; Huang, Y.B.; Wu, P.C.; Tsai, Y.H. 1996. Transdermal iontophoresis of sodium nonivamide acetate II: optimization and evaluation on solutions and gels. Int. J. Pharm.145, 175-186.
2.
3. 4. 5.
6.
7.
8.
9.
10. Salem H. Spectrophotometric determination of beta-adrenergic blocking agents in Pharmaceutical formulations. J. Pharm. Biomed. Anal. 2002, 29, 527-538. 11. Bhaskaran, S., Harsha, N. S. 2000. Effect of permeation enhancer and iontophoresis on permeation of atenolol from transdermal gels. Ind. J. Pharm. Res. 6, 424-426. 12. Al-Ghannam S.M.; Belal F. Kinetic Spectrophotomtric determination of atenolol in dosage forms. J AOAC Int. 2002, 85, 817-823. 13. Vetuschi C.; Ragno G. Fourth UV derivative spectrophotometry for the simultaneous assay of atenolol and chlorthahlidone in pharmaceuticals. Int. J. Pharm. 1990, 177-181.
Conclusion
It is thus concluded that the proposed method is new, simple, and also accurate, cost effective, and precise and not so time consuming. The proposed method can be successfully employed in the routing analysis of Atenolol in gel formulations which are generally used for iontophoresis delivery system as effective novel transdermal preparations.
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UV-Spectrophotometric Estimation of Nabumetone$

M. N. Purohit *, Sharad Mohan, Kunal Chokshi, G. V. Pujar
Abstract: A simple and sensitive spectrophotometric method has been developed for determination of nabumetone in bulk powder and its tablet dosage form. The method is found to be linear over the concentration range of 10-35 g/ml. for method A and 8-25 g/ml. for method B with a regression coefficient of 0.9994 and 0.9978 respectively. The method was validated and can be used successfully to assay nabumetone in bulk powder and its tablet dosage form.
Introduction
Nabumetone is a Non-Steroidal anti-inflammatory and analgesic drug. Chemically Nabumetone is 4-(6-methoxy-2-napthalenyl)-2butanone.1 It is official in US pharmacopoeia and British pharmacopoeia.
O CH3 H3CO
Aliquots of 0.2ml to 3.5ml portion of standard solutions were transferred to a series of 10ml volumetric flask and further diluted with acetonitrile upto the mark. The absorbance of the solution in each flask was measured at 271 nm against the reagent blank and calibration curve was plotted. Similarly the absorbance of sample solution was measured and the amount of nabumetone was determined by referring to the calibration curve. Method B: A standard solution containing 1 mg/ml Nabumetone was prepared by dissolving 100mg of pure drug in 100ml of mixture of acetone: methanol (10:90). It was further diluted with methanol to obtain 100 g/ml concentrations. Aliquots of 0.2ml to 2.5ml portion of standard solution were transferred to a series of 10ml volumetric flask and further diluted with methanol up to the mark. The absorbance of the solution in each flask was measured at 254 nm against the reagent blank and calibration curve was plotted. Similarly the absorbance of sample solution was measured and the amount of nabumetone was determined by referring to the calibration curve.
In the literature only HPLC methods are available.2-3 There are no spectrophotometric methods reported hitherto. Hence it was thought to develop simple spectrometric methods to estimate Nabumetone in routine bulk drug sample and tablets. The present work describes two simple ultra violet spectrophotometric methods for Nabumetone using solvents such as acetonitrile (method A) and mixture of acetone and methanol (method B).
Materials & Methods

Shimadzu UV-Visible spectrophotometer-1601 was used for the analysis. Nabumetone sample was obtained as a gift sample from Divis lab. Hyderabad. Tablets of nabumetone were purchased from local market. All other solvents and reagents were of AR grade.
Method of Preparation of Sample

Twenty tablets were weighted and powdered. The powder equivalent to 50mg of nabumetone was accurately weighed and dissolved in acetonitrile for method A and mixture of acetone and methanol (10: 90) for method B. (0.5 mg/ml). From this solution further dilutions were made to obtain 5g/ml concentration. The absorbance of the sample was measured at appropriate wavelength. The optical characteristics and precision are presented in Table 1. Percent recovery study were carried out by adding a known amount of nabumetone to the sample.
Preparation Of Standard
Method A: A standard solution containing 1 mg/ml Nabumetone was prepared by dissolving 100mg of pure drug in 100ml of acetonitrile. It was further diluted with the same solvent to 100 g/ml.
Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Mysore, Karnataka

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Tabel 1: Optical Charactristics And Precision Parameters g max (nm) Beer's law limit (g/ml.) Correlation coefficient (R ) Slope Molar absorptivity % Recovery
2
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Conclusion
Method B 254 8-25 0.9978 0.0282 Two simple and sensitive uv-spectrophotometric methods were proposed for the estimation of nabumetone. Both methods gave reproducible result for the estimation of the drug in bulk powder and the tablet dosage form. The methods can be employed for the routine analysis in the pharmaceutical industries.
Method A 271 10-35 0.9994 0.0204 2.6 X 10

3
8.7 X 103 99.26%
Acknowledgements
We sincerely thank Principal and Head, Dept of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Mysore for encouraging us to carry out this work. Authors are also thankful to Divis lab. Hyderabad for the gift sample of nabumetone.
99.13%
Result & Discussion

Beer Lambert's law is obeying in the concentration range of 10-35 g/ml for method A and 8-25 g/ml for method B. The linearity of standard plot of Nabumetone was found to be 0.9994 for method A and 0.9978 for method B. The percentage recovery for Nabumetone is found to be 99.13% by method A and 99.26% by method B. In addition the peak pattern of standard and sample were same, which indicates that there was no interaction or interference of excipients.
References
1. Robert E. Willette, Analgesic Agents, Wilson and Griswold Text book of Organic Medicinal and Pharmaceutical chemistry, , 11th edition, Lippincott Williams & Wilkins, 2004, 759. United States Pharmacopoeia, XXIII, NF XVIII, The United States Pharmacopoeial Convention Inc., Rockville, MD, 2004, 1268. British pharmacopoeia, Ministry of Health & Family Welfare, H.M. Stationary Office, London, 1996, 1808.
2. 3.
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Difference Spectroscopic Estimation of Raloxifene in Bulk and Formulations

Patel P. M., Patel R. C.* and Patel N. M.
Abstract: A simple and highly sensitive difference spectroscopic method for the determination of Raloxifene in bulk and dosage form is described. The proposed method is based on the principle that Raloxifene can exhibit two different forms in acidic and basic medium that differs in their absorption spectra in acidic and basic medium. The maxima in difference spectrum are at 310 nm, and minima at 285 nm. The proposed method can be successfully used for the analysis of drug in marketed preparations in the range of 10-60 g/ml and minimum concentration measured is 1 g/ml. Raloxifene concentration in g range can be estimated by this method. The method is found to be rapid, precise and accurate and can be applied for routine estimation of Raloxifene.
Introduction
Raloxifene hydrochloride (RLH), [6-hydroxy-2-(4-hydroxy phenyl) benzo[b]thien-3-yl]-[4-[2-(1-piperinyl) ethoxy]-phenyl] methanone, is an antiosteoporotic. It is a nonsteroidal benzothiophene, which is the first selective oestrogen receptor modulator (SERM) to be approved for the prevention and treatment of osteoporosis in postmenopausal women. It is listed in Merck Index. 1 A survey of literature revealed a spectrophotometric,2 capillary electrophoresis3 and a few chromatographic methods for its determination in bulk drug4 and in plasma.5 No method has been so far reported for the estimation of RLH from pharmaceutical dosage forms.
to prepare working solution of 100 g/ml. The absorbance of working solutions were measured with reference to blank and the amount of Raloxifene was calculated from calibration curve, and
Table 1: Results of The Marketed Raloxifene Tablets
Formulation T1 T2 mg of drug Amount found % Labeled claim taken from tablets in (mg) *Meansd 5 5 4.95 4.95 99.30.38 99.30.38
* Average of three determinations T 1 Bontact 60 mg manufactured by Cadila Pharma Ltd. T 2 Ralofen 60mg manufactured by Lupin Lab Ltd.
Material Methods
In the present investigations, a new simple selective and sensitive different spectrophotometric method is reported for Raloxifene in bulk drug formulations. Standard solutions were prepared by dissolving Raloxifene in methanol to make final concentration of 100 g/ml as a stock solution. Different aliquots were taken from stock solution diluted with buffer solution of 0.1 N HCl and 0.1 N NaOH separately to prepare series of concentration 10-60 g/ml as a reference and test solutions respectively. Maxima and minima were obtained as 310 nm and 285 nm. The calibration curve was prepared by plotting amplitude versus concentration of Raloxifene. Tablet of two brands were used for the purpose of analysis. 20 tablets were powdered and powder equivalent to 10 mg was transferred in volumetric flask and dissolved in 0.1 N HCl to prepare working solution of 100 g/ml. Similarly was taken another sample of 10mg of Raloxifene dissolve it in 0.1 N NaOH
the results are shown in Table 1.

Most of the formulations having excipients and binders along with active drug constituents. These excipients may cause some interference during the estimation of active constituents. Interference or absence of interference was confirmed by performing the recovery experiment, for which standard addition method was employed. From the recovery results it is claimed that the method can be used for estimation of Raloxifene in dosage form. The results are shown in Table 2. Raloxifene showed maxima and minima at 310 nm and 285 nm by respectively taking drug solution in 0.1 N HCl in reference cell and drug solution in 0.1 N NaOH in test cell. The calibration curve was found to be linear in range of 10-60 g/ml. From the results shown in Table 1, it is concluded that the excipients present in the formulation do not interfere in the estimation of Raloxifene. This method is also suitable for estimation of its dosage form. This
Shri B. M. Shah College of Pharmaceutical Education and Research Modasa. Guj. Author for correspondence: E-mail: raks.rids@gmail.com
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Table 2: Optical and Statistical Data Parameters Maximum wavelength max Minimum wavelength min Calibration curve range Molar extinction coefficient Scandell's sensitivity Regression equation Slope Intercept Correlation co-efficient (r)
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Values 310nm 285nm 10-60 g/ml 3.455 x 104 0.0117 g/ml Y= 0.0706X+0.094 0.0706 0.094 0.995
method can be applied to marketed preparations. The slop, intercept, co-relation coefficient and optical characteristics are summarized in Table 2. The proposed method can be successfully used for the analysis of drug marketed preparations with good precision, sensitivity and accuracy.
References
1. 2. 3. 4. 5. Budavari. S., Eds., In; Merck Index, 13th Edn., Merck & Co., Inc., Whitehouse Station, NJ, 2001, 1452. Dharuman, J., Ravichandran, V., Thirumoorthy, N and Dharamsi, A., Pharmazie, 2004, 59,720. Perez-Ruiz, T., Martinez-Lozano, C., Sanz, A and Bravo, E., J. Pharm. Biomed. Anal., 2004, 34, 891. Nandini, P and Jayant,W.P., Indian Drugs., 2001, 28, 591. Zweignenbaum, J and Henein, J., Anal. Chem., 2000, 72, 2446.
Source: The Pharma Review, April 2007
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Spectrophotometric Methods for the Determination of Rosuvastatin Calcium in Pure Form and in Pharmaceutical Formulations by Using Redox/Complexation Reactions
M. Vamsi Krishna and D. Gowri Sankar*
Abstract: Three simple, sensitive and cost effective Spectrophotometric methods are described for the determination of rosuvastatin calcium (RST) in pure form and in pharmaceutical formulations. These methods are based on the oxidation of RST by ferric chloride in presence of o-phenanthroline (Method A) or 2, 2' Bipyridyl (Method B) or potassium ferricyanide (Method C). The colored complex formed was measured at 520, 530 and 750nm for method A, B and C respectively against the reagent blank prepared in the same manner. The optimum experimental parameters-for the color production are selected. Beer's law is valid with in a concentration range of 5-25g ml-1 for method A and 3-15g ml-1 for method B and C. The mean percentage recoveries are 99.71, for method A, 100.01 for method B and 99.06 for method C. The developed methods are applied for the determination of RST in bulk powder and in the pharmaceutical formulations without any interference from tablet excipients.
Introduction
Rosuvastatin calcium (RST) is an anti hyperlipidimic agent and is chemically known as 6-heptenoic acid, 7-[4-((4-fluorophenyl)-6(1-methylethyl)-2-[methyl (methylsulphonyl) amino]-5pyrimidinyl]-3, 5-dihydroxy-, calcium salt (2:1), (3R, 5S, 6E). It acts by inhibiting the enzyme 3-hydroxy-3-methyl glutarylcoenzyme A (HMG-coA) reductase. To the best of our knowledge, there is no work in the literature reported about the visible spectrophotometric methods for the quantification of RST in pure 1 drug and in pharmaceutical formulations, however one HPTLC 2-4 and few LC-MS methods have been reported for the estimation of RST in pharmaceutical preparations and in biological fluids. The proposed methods are based on the oxidation of drugs by Fe (III) in presence of o-phenanthroline or 2,2 bipyridyl or potassium ferricyanide. The colored complex formed was measured at 520, 530 and 750 nm for method A, B and C respectively.
Materials and reagents: All chemicals used were of analytical reagent grade. RST was obtained from Dr. Reddy's Labs Hyderabad. Fortius, Rostar and Novostat are the commercial tablet formulations labeled to contain 5, 10 and 20mg of RST per tablet respectively. o-phenanthroline (0.2%) was prepared by dissolving 200mg of ophenanthroline in 100 ml of distilled water. Ferric chloride (0.5%) was freshly prepared by dissolving 500mg of ferric chloride in 100ml of distilled water. 0.02M orthophosphoric acid was prepared by mixing 8.5ml of orthophosphoric acid with distilled water and final volume make up to 1000ml potassium ferricyanide (0.1%) was prepared by dissolving 100mg of potassium ferricyanide in 100 ml of distilled water. Stock reference solution (100g ml ) was freshly prepared from pure sample of RST by dissolving 0.01g in 100ml of solvent (Methanol and water in the ratio of 1:1).
-1
Experimental
Apparatus: All spectral and absorbance measurements were made on a systronic model 106 digital spectrophotometer with 10mm matched quartz cells.
General Procedure and Calibration

In methods A and B, different aliquots of stock reference solution -1 (100g ml ) from 0.5-2.5 ml (for method A) or 0.3-1.5 ml (for
Pharmaceutical Analysis and Quality Assurance Division, Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam. For correspondence author: E-mail: gowrisankar97@rediffmail.com
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method B) were transferred in to a series of 10 ml standard flasks. To each flask 1.5ml of ferric chloride and 1.5 ml o-phenanthroline o or 2, 2 bipyridyl were added and kept in a water bath (601 c) for o 15 min, then immediately cooled to room temperature (251 c) using a cold water bath and I ml of o-phosphoric acid was added. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 520nm (method A) or 530nm (method B) against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation. In method C, different aliquots of stock reference solution (100g -1 ml I) from 0.3-1.5 ml were transferred in to a series of 10 ml standard flasks. To each flask 0.5ml of ferric chloride and 0.5 ml potassium ferricyanide were added. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 750nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.
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upper beers law limits. The measured standard deviation (S.D.), Relative standard deviation (RSD), and confidence limits can be considered satisfactory for all the three methods and are summarizes in Table 1.
Table 1: Optical and Regression Characteristics, Precision and Accuracy of the Proposed Methods for RST Parameter lmax (nm) Beers law limits (g ml ) Detection limits (g ml ) Molar absorptivity (L mole cm ) Sandells sensitivity (m cm-2 / 0.001 absorbance unit) Optimum photometric range (g ml-1) Regression equation (Y = a + bC) Slope (b) Standard deviation-of slope (Sb) Intercept (a) Standard deviation, of intercept (Sa) Standard error of estimation (Se) Correlation coefficient (r) Relative standard deviation (%)* %Range of error (Confidence limits)* 0.05 level 0.01 level 0.187 0.277 0.118 0.175 0.150 0.221 0.136 3.14x10-2 0.90x10-4 2.00x10-4 1.53x10-3 1.46x10-3 0.9999 0.223 5.45x10-2 1.00x10-4 1.00x10-4 9.90x10-4 9.50x10-4 0.9999 0.141 6.11x10-2 1.70x10-4 5.00x10-2 1.66x10-3 1.58x10-3 0.9999 0.178
-1 -1 -1 -1
Method A Method B Method C 520 5.0-25.0 0.038 3.15x10 0.031 6.0-23.0

4
530 3.0-15.0 0.031 5.46x10 0.018 4.0-13.0

4
750 3.0-15.0 0.510 6.11x104 0.016 4.0 -13.0
Procedure for Tablets

Twenty tablets were weighed accurately and ground in to a fine powder. An amount of powder equivalent to 10 mg of RST was weighed into a 100ml volumetric flask, 50 ml of the solvent was added and shaken thoroughly for about 10 min, then the volume was made up to the mark with the solvent, mixed well and filtered using a quantitative filter paper. The assay of the tablets was completed according to the general procedure.
% Error in bulk samples** 0.099 0.229 * Average of eight determinations * * Average of three determinations In Y=a+bC, Y is absorbance and C is concentration.

Methods A, B and C are based on the oxidation of RST by excess + of ferric salt (Fe III or Fe 3 ) and the reduced state of FeIII was utilized besides the unreacted FeIII. The Fe II has tend ency to give colored complex on treatment with o-phenanthroline or 2,2' bipyridyl or potassium ferricyanide. As Fe III interfere even though to a little extent (especially in the lower range of beer's law limits) in the determination of Fe II in methods A and B, the reactivity of the interfering entity (Fe III) has to be made insignificant by complexing it with o-phosphoric acid. Optical characteristics such as Sandell sensitivity, molar absorptivity, precision and accuracy were found by performing eight replicate determinations containing 3/4th of the amount of the
Application in Pharmaceutical Analysis and a Statistical Comparative Study

The proposed methods (A, B and C) were applied to the spectrophotometric determination of RST in commercial pharmaceutical formulations. The results obtained were compared statistically by the student's t-test and the variance ratio F-test with those obtained by applying the UV spectrophotometric method for RST developed in our laboratory on samples of the same batch and given in Table 2.
Conclusion
The proposed methods are simple, accurate and offer advantages of reagent availability and stability, less time
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Table 2: Results of analysis of tablet formulations containing RST Method Forumlation Labeled % recoverya % recoverya anount of reference (mg) methodd A Fortius Rostar Novostat Fortius Rostar Novostat Fortius Rostar Novostat 5 10 20 5 10 20 5 10 20 99.71 100.05 100.20 100.01 101.15 101.35 100.20 101.35 100.08 99.98 100.35 100.10 99.98 100.35 100.10 99.98 100.35 100.10 tb Fc
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the determination of RST in pharmaceutical formulations without interference from excipients such as starch and glucose and from common degradation products, suggesting applications in bulk drug analysis
0.27 10.7 0.96 1.12 1.01 1092 1.25 1.31 1.54 1.97 0.21 1.21 3.01 1.22 2.10 2.58 2.43 2.22
References
1. Sane, R.T.; Kamat, S.S.; Menon, S.N.; Inamdar; Shafi, R.; Mander, R.Determination of rosuvastatin calcium in its bulk drug and pharmaceutical preparations by HPTLC. Journal of planar chromatography-Modem TLC. 2005,18 (103),194-198. Kathalijne, A.; Oudhoff; Timothy Sangster; Elizabeth Thomas; Ian, D.; Wilson. Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma. J. Chromatogr. B. 2006,832(2), 191-196. Ravi Kumar, T.; Raja Reddy, K.; Ramesh, M.; Srinivas, R.; Simultaneous,determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS "" With electrospray ionization: Assay development, validation and application to a clinical study. J. Pharm. Biomed. Anal.2005, 39(3-4), 661~669. Hull, C.K.; Penman, A.D.; Smith, C.K.; Martin, P .D. Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences.2002. 772 (2), 219-228.
2.
C
a b
Average of six determinations Calculated t-value; tabulated t-value for five degrees of freedom; and p=0.05 is 2.57 Calculated F-value; tabulated F-value for five degrees of freedom; and 95% confidence limits is 5.05. UV method developed in our laboratory.
3.
4.
consumption and high sensitivity. Although in methods A and B, the color development at room temperature requires 2 hr for completion, this can be shortened to 15 min by raising the temperature to 605oc. The proposed methods are suitable for
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A Novel Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Frusemide in Tablets

R. K. Maheshwari Abstract: Several methods are used to increase the aqueous solubilities of poorly water-soluble drugs. Hydrotropic solubilization technique is one of them. In the present investigation, hydrotropic solution of sodium benzoate (2.0 M) has been employed as solubilizing agent to solubilize poorly water-soluble drug, frusemide, from fine powder of its tablets for spectrophotometric determination in ultraviolet region. Frusemide shows maximum absorbance at 330 nm. Beer's law was obeyed in the concentration range of 20 to 120 g/ml. Results of analysis were validated statistically and by recovery studies. The proposed method is new, accurate, simple, cost-effective, environmentally friendly and can be successfully employed in routine analysis of frusemide tablets. Sodium benzoate did not interfere in estimation.
Introduction
The term "hydrotropy" has been used to designate the increase in solubility of various substances due to the presence of large amount of additives. Various hydrotropic agents have been used to enhance the aqueous solubility of a large number of drugs.1-14 Maheshwari has developed new analytical methods based on hydrotropic solubilization phenomenon for poorly water-soluble drugs cefixime,1 ketoprofen,2,3 salicylic acid,3 tinidazole,4 aceclofenac,5 norfloxacin6 and amoxycillin.7 Maheshwari et al have analyzed poorly water-soluble drugs ofloxacin, 8 hydrochlorothiazide,9 metronidqazole,10 tinidazole,10 nalidixic acid,10 norfloxacin,10 ibuprofen,11 naproxen11 and flurbiprofen11 using hydrotropic solubilization phenomenon. C h e m i c a l l y, f r u s e m i d e ( 4 - c h l o r o - N - f u r f u r y l - 5 sulphamoylanthranilic acid) is a widely used diuretic drug. There was tremendous increase in aqueous solubility of frusemide in 2.0 M sodium benzoate solution (more than 90 fold enhancement in aqueous solubility as compared to solubility in distilled water). Therefore, it was thought worthwhile to solubilize frusemide to carryout spectrophotometric estimation. Sodium benzoate did not interfere in spectrophotometric estimation.
Materials Gift sample of frusemide was supplied by Alkem Laboratories Limited, Mumbai. Commercial tablets of frusemide were purchased from local market. All other chemicals and solvents were of analytical grade. Calibration curve About 0.1 g frusemide was accurately weighed and transferred to a 100 ml volumetric flask. Twenty ml of 2.0 M sodium benzoate solution was added and drug was solubilized in it by shaking and volume was made upto 100 ml with distilled water. By appropriate dilution with distilled water, the standard solution (200 mg/ml) was obtained. The standard solution was further diluted with distilled water to obtain various dilutions 20, 40, 60, 80, 100 and 120 mg/ml of frusemide. Absorbances of these solutions were measured at 330 nm against corresponding reagent blanks. A linear relationship was observed over the range of 20 to 120 mg/ml. Preliminary solubility studies of frusemide Solubility of frusemide in distilled water and 2.0 M sodium benzoate solution were determined at 2810 C. Enhancement in the solubility of frusemide in 2.0 M sodium benzoate solution was more than 90 folds (as compared to its solubility in distilled water). Analysis of frusemide tablets by the proposed method Tablet powder equivalent to 0.1 g of frusemide was transferred to a volumetric flask of 100 ml capacity containing 20 ml of 2.0 M
Experimental
Instrument The instrument used was Shimadzu UV/Visible recording spectrophotometer (model UV-160 A) with 1 cm matched silica cells.
Department of Pharmacy, S.G.S.I.T.S., Indore. E-mail rkrkmaheshwari@indiatimes.com

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sodium benzoate solution. The flask was shaken for 5 minutes to solubilize the drug and then volume was made upto the mark with distilled water. Solution was filtered through Whatman filter paper no. 41. Filtrate was divided in two parts, A and B. Part A was kept at room temperature for 48 hours to check the effect on stability of drug in presence of sodium benzoate and also to note precipitation, if any, during this period. Filtrate of part B was appropriately diluted with distilled water and absorbance was noted at 330 nm against reagent blank and drug content was calculated (Table 1). After 48 hours, filtrate of part A which had no evidence of precipitation was appropriately diluted with distilled water and analyzed for drug content. Table 1: Results of analysis of commercial tablets of frusemide
Tablet Label claim % Label claim formulation (mg) estimated* (mean S.D.) I II III 40 40 40 100.38 1.223 99.09 0.944 100.68 0.882 % Coeff. Standard of variation error 1.218 0.953 0.876 0.706 0.545 0.506
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The solubility of frusemide in 2.0 M sodium benzoate solution was found to be more than 90 fold as compared to its solubility in distilled water. Thus it was thought worthwhile to solubilize the drug present in fine powder of its tablets to carryout spectrophotometric estimation. The percent label claims estimated using the proposed method ranged from 99.09 0.944 to 100.68 0.882 and recovery being 98.82 - 101.33%
Conclusion
Methanol, ethanol, chloroform, carbon tetrachloride and acetone find wide use in spectrophotometric estimations of poorly watersoluble drugs. Most of these organic solvents are toxic, costlier and sources of pollution. Inaccuracy in spectrophotometric estimations due to volatility is another drawback of these volatile solvents. In the present investigation frusemide was used as a model drug having poor aqueous solubility. Sodium benzoate did not interfere in the spectrophotometric estimation. The proposed method can be successfully employed in the routine analysis of frusemide in tablet dosage forms. Source: The Pharma Review, October 2006
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Estimation of Fenoverine Hydrochloride in Pharmaceutical Dosage Forms by RP HPLC

Dr. K. Chitra*, K. Sujatha, C. Vinodhini, B. Hareesh, B. Bharath, E. Brahma Reddy and B. Pamula Reddy
Abstract: A simple, precise and reproducible method has been developed and validated for the estimation of Fenoverine Hydrochloride in pharmaceutical dosage forms by RP HPLC. Literature survey revealed that no method has been reported for the estimation of Fenoverine Hydrochloride except its determination in human plasma. The separation was achieved using C-18 ODS/RP column (5 micron/25 cm x4.6 mm) in isocratic mode with mobile phase ammonium acetate and acetonitrile (50:50 v/v) at a flow rate of 1.5 ml/min, detection was carried out at 254 nm using a UV detector. The retention time was 5.5 min. Dilution of standard and sample were made in mobile phase. Recovery study was carried out by adding known quantity of standard drug in the preanalysed test solution and % recovery is calculated in each case. The percentage recovery study of Fenoverine Hydrochloride was found to be 98.5 to 100.1% w/w respectively. The proposed method was found to be accurate, precise, simple and rapid which could be easily used for routine analysis.
Introduction
Fenoverine Hydrochloride is a non anticholinergic synchronizer of smooth muscle motility.1 It is being used for the treatment of irritable bowl syndrome and urogenital tract (egdysmennorrnea). It is chemically2 10 [4-piperonyl 1-piperazinyl) acetyl] phenothiazine, having the molecular formula C26H25N3O3S. It is not official in any pharmacopoeia. Literature survey reveals that no specific method has been reported for the estimation of Fenoverine Hydrochloride individually or in combination of other drugs .However only one HPLC method3 has been reported for the analysis of Fenoverine Hydrochloride in biological fluids. This paper presents a simple accurate reproducible rapid and economic method for the estimation of Fenoverine Hydrochlorides in pharmaceutical dosage forms.
Standard stock solution

Accurately weighed 100 mg of Fenoverine Hydrochloride working standard was dissolved in 50 ml of ethanol in 100 ml of volumetric flask and diluted to the mark (1 mg Fenoverine Hcl ml1) and filter through 0.45 filter paper. (1 mg ml-1)
Sample preparation
20 capsules were weighed and mixed to fine powder. A powder equivalent to 100 mg of Fenoverine Hydrochloride was weighed accurately and extracted with ethanol and volume made up to 100ml with ethanol. The solution was filtered through 0.45 filter paper.
Chromatographic conditions
Mode of separation Column : Reversed phase -Isocratic : RPC-18, ODS/RP column (5 ) (250 x 4.0 mm pre washed with ethanol ( 15 minutes) prior to analysis : 500 ml of ammonium acetate: 500 ml of acetonitrile were mixed and filtered through 0.45 filter paper. : 1.5 ml/min : 1.0 : ambient.
Experimental Conditions
High performance liquid chromatograph Shimadzu HPLC VP series LC 10 ATVP SPD 10 AVP Rheodyne injector with 20 l loop and UV- Detector Mobile phase
Chemicals and reagents

Working standard of Fenoverine Hydrochloride, Ammonium acetate of AR grade and acetonitrile of HPLC grade. Mobile phase: 500 ml acetonitrile: 500 ml ammonium acetate were mixed and filtered through 0.45 l filter paper.
Flow rate Chart speed Temperature
Detector wave length : U .V 254 nm.
*Author for correspondence: Professor, Sri Ramachandra College of Pharmacy, SRMC & RI (DU), Porur, Chennai. Email: chitrasrmc@rediffmail.com
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ANALYTICAL METHOD DEVELOPMENT Procedure

The column was flushed with mobile phase till stable base line obtained.20 l of standard and sample dilution were injected separately under the chromatographic condition described above and scans were recorded Each solution was run 5 times. The amount of Fenoverine Hydrochloride in sample was calculated by companies the peak area ratio from standard.
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Recovery
To establish the accuracy reproducibility and precision of the proposed method, recovery experiments were carried out by spiking the pre- analyzed sample at three different levels. The results are recorded in table 1 below. Table 1: Estimation of Fenoverine Hydrochloride in Dosage Formulation. S. No 1. 2. 3. Claim(mg) 100 100 100 Fenoverine Hydrochloride Found (mg*) Recovery (%)** 101.22 98.53 101.58 100.1 99.08 100.3
Au 0.63
-0.16
2.42
0.00
4.91
5.54
Fenoverine Hydrochloride was estimated with retention time of 5.5 minutes and the linearity was found in the range of 0.01 mg/ml to 0.05 mg/ml. The chromatogram was scanned at 254 nm, being the absorption maxima of Fenoverine Hydrochloride. The assay value and recovery data indicate that the method is free from interference of excipients and can be effectively used for routine 1.41 quality control.
win
9.83
Typical HPLC chromatogram of sample containing Fenoverine Hydrochloride.
References
1. 2. 3. Chitra K., Seenivasan P.,Vinodhini C., Vasantha Janardhan .,The Antiseptic., 2005, 102 (3),117-119 Martindale, The Extra pharmacopoeia, 32nd edition, The pharmaceutical press, 1999, London, 1579-2. Hu OY, Chen PH, Fang YJ, Tang HS, PaoLH, Kwok KM, Kind ML, J.Pharm Sci, 1992, 81(1) 91-93.
*Average of three determination **After spiking the pre analysed sample at different levels.

Under proposed chromatographic conditions, the drug
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Evaluation of Marketed Polyherbal Antidiabetic Formulations using Biomarker Charantin

P. M. Patel1, N. M. Patel1 and R. K. Goyal* Abstract: Charantin is one of the phytoconstituents present in Momordica charantia Linn. M. charantia is known for its hypoglycemic activity from ancient times. In the present study an attempt has been made to develop a HPTLC method for quantitative estimation of charantin in small, big, fresh fruits and different marketed antidiabetic polyherbal formulations (PHF). Silica gel 60 F254 precoated plates (10 x 10 cm) were used with benzene: methanol (80:20) as solvent system. Sample was spotted on precoated TLC plates by using Linomat 5 spotter. Ascending mode was used for development of thin layer chromatography. TLC plates were developing up to 8 cms. The plates were sprayed with 10% sulphuric acid in alcohol and the reagent must be prepared freshly, heated at 1300 C for 2-3 min and brought to room temperature. Violet spot with Rf 0.32 was visible and scanned under 536 nm. This HPTLC method was found to be reproducible, accurate and precise and could detect charantin concentration at nanogram level. The developed HPTLC method would be an important tool in the way of acceptability of quality control method of polyherbal formulations. Data revealed that phfl contained highest quantity of the charantin. We found little less amount of charantin in phfd and phfj. It may be due to varied reasons like time of collections, age of the pant, processing conditions, incorrect identification of the plant, improper selection of the herb variety, addition of exhausted material and genetic variety of the plat material.
Introduction
Diabetes mellitus has recently been identified by Indian council of medicine research (ICMR) as one of the refractory diseases for which satisfactory treatment is not available in modern allopathic system of medicine and suitable herbal preparations are to be investigated.1 WHO has approved the use of traditional medicines as part of health programme. A herbal medicine is defined as a finished, labeled medicinal product that contains active ingredients as aerial or underground parts of plants or other plant material or combinations thereof. Approximately 3300 million people in the underdeveloped countries use medicinal plants on a regular basis while there has been great fascination for the herbal medicines and dietary food supplements in the developed countries.2 India has a rich heritage of usage of medicinal plants in the Ayurvedic, Siddha and Unani system with a mention of about 45,000 plants.3-4 A large number of plant preparations have been reported to possess antidiabetic activity over last several decades.5 A database of natural hypoglycemics collected by researchers in Mexico lists almost 800 plants. Researchers in India have documeted the use of over 150 plants in families with reported hypoglycemic activity.6 A recent crosscultural compendium over cites 1,200 medicinal plants used for diabetes.7 There are around 6000 herbal manufacturers in India.
1
More than 4000 units are producing Ayurvedic medicines. Due to lack of Infrastructure, skilled manpower reliable methods and stringent regulatory laws most of these manufacturers produce their products on very tentative basis. Herbal drug formulations have received a raw deal from the modern medicine due to many reasons. Diabetes is a heterogeneous chronic metabolic disorder characterized by hyperglycemia resulting from defect in insulin secretion and / or deficiency of insulin secretion. Large numbers of polyherbal formulations are available and being prescribed Nation wide even by registered doctors for diabetes mellitus. The rational for the use and compliance are either not available or known for these herbal formulations. Majority of herbal drugs are multiherbal combinations, whose rationality is yet to be proved in all the cases. Standardization is a essential measurement for ensuring the quality control of the herbal drugs. Momordica charantia Linn. Cucurbitaceae is a well known to possess antihyperglycemia, anticholesterol, immuno suppressive, antiulcerogenic, anti sepermatogenic and androgenic activities.8-10 One of the active principle reported to be responsible for various actions is charantin.11-13 All the three selected polyherbal formulations contain karela as one of the plants. The reference standard charantin is not available commercially, charantin had to be isolated, purified and the
Shri B.M. Shah College of Pharm. Educ. & Res., Modasa. *Dept. of Pharmacology, L.M. College of Pharmacy, Navrangpura, Ahmedabad. *For correspondence: goyalrk@hotmail.com
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structure authenticated by various spectral analysis. There are no titrimetric, colorimetric, spectrophotometric or chromatographic methods available for quantitative estimation of charantin in different marketed antidiabetic PHFs. Therefore an attempt has been made to develop a HPTLC method is fast, precise, sensitive and reproducible with good recoveries for standardization of polyherbal formulations.
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Material and Methods

Equipment: A camag HPTLC system equipped with a sample applicator Linomat V, twin trough plate development chamber, TLC Scanner III and Wincats an integration software 4.02 (Switzerland). Chemicals: Analytical grade chloroform, benzene, methanol, formic acid ethyl acetate were obtained from S.D. Fine Chem Ltd (Mumbai, India). TLC aluminium plates pre-coated with silica gel 60 F254 (10x 10 cms, 0.2 mm thick) used were obtaind from E. Merck Ltd (Mumbai, India). Sample preparation: Three polyherbal formulations were taken for the quantitative estimation of biomarkers like charantin, curcumin and swetiamarin. The selected PHFs were Mersina (PHFj, capsule), Diabecone (PHFd, tablet) and Madhuripu (PHFl, powder). 200 mg of these selected PHFs were taken for the quantitative estimation. HPTLC method for estimation of charantin: Preparation of calibration curve of standard charantin: One milligram of working standard charantin was dissolved in 100 ml of chloroform to yield stock solution of 100g/ml concentration. Calibration curve from 20-600ng/spot was prepared and checked for reproducibility, linearity and validating the proposed method. The correlation coefficient, coefficient of variance and the linearity of results were calculated. Sample preparation: 200 mg of PHfs were taken and extracted in 10 ml of chloroform then the chloroform extract was filtered through Whatmann no. 42 filter paper. The final volume of the extract was made to 10ml with chloroform in volumetric flask Charantin contents was analysed after subjecting to HPTLC. The small karela, big karela and fresh karela were dried under shade and finely powdered. From that 50 mg of fine powder was taken and extracted by chloroform filtered it, dried it and finally make the volume of the extractives up to 2 ml with chloroform. Method Specifications: Silica gel 60 F254 precoated plates (10 x 10 cm) were used with benzene: methanol (80:20) as solvent system. Sample was spotted on precoated TLC plates by using Linomat 5 spotter. Ascending mode was used for development of
thin layer chromatography. TLC plates were developing up to 8 cms. The plates were sprayed with 10% sulphuric acid in alcohol and the reagent must be prepared freshly, heated at 1300 C for 2-3 min and brought to room temperature. Violet spot with Rf 0.32 was visible and scanned under 536 nm. The contents of charantin in the selected PHFs were determined by comparing area of the chromatogram of PHFs with calibration curve of the working standard of charantin.
AU 300 250 200 150 100 50 0 -0.03
Track 6.ID: charantin
Charantin
-0.17
-0.37
-0.57
-0.77
-0.97
Rf
Fig.1: HPTLC chromatogram of standard charantin

Sample Small Karela Big karela Fresh fruits Phf L Phf D Phf J Amount of charantin per ng/spot 301 235.88 193.15 375.21 257.68 252.18 %w/w charantin 0.602 0.4716 0.3863 0.09356 0.06443 0.06305
Table 1: Percentage of Charantin in different type of M.charantia fruits and its formulations by measuring area in HPTLC method

Validation of HPTLC method: 1. Linearity: A representative calibration curve of charantin was obtained by plotting peak area of charantin against the conc4ntration of charantin over the range of 20- 600 ng/spot. The correlation coefficient was found to be 0.9635. 2. Accuracy (% Recovery): The % recovery of charantin given in table 3 was found to be 98.89 which is satisfactory. 3. Specificity: It was observed that the other herbs present in the formulations their constituents and excipients did not interfere with the peak of charantin. Therefore the method was specific. The spectrum of standard charantin spot and charantin in other sample was found to be similar or overlap.
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4. Limit of Detection: The minimum detectable limit was found to be 20ng / spot. Standard charantin showed single peak in HPTLC chromatogram. The calibration curve of charantin was obtained by spotting standard charantin on HPTLC plate. After development the plate was scanned at 536 nm (fig.1). The calibration curve was prepared by plotting the concentration of charantin versus average area of the peak . PHFs were analysed by the proposed method. The amount of charantin was computed from calibration curve. Data revealed that phfl contained highest quantity of the charantin. We found little less amount of charantin in phfd and phfj. It may be due to varied reasons like time of collections, age of the pant, processing conditions, incorrect identification of the plant, improper selection of the herb variety, addition of exhausted material and genetic variety of the plat material.
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found to be about 98.89%, which shows the reliability and suitability of the method. The lowest detectable limit of charantin in different formulations was found upto 20 ng/spot.
References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Palimbo, P.J., Diabetes, 1976, 25, 566. Dobriyal, R.M. and Narayana, D.B., IDMA Bulletin, 1998, 29, 272. Hakim, Z.S., Bangaru, R.A., Santani, D.D. and Goyal R.K., Ind. J. Nat. Prod., 1995, 11, 3. Deshmukh, P.G., IDMA Bulletin, 1998, 29, 228. Bever, B.O. and Zahand, G.R., Crude Drug Res,, 1979, 17, 139. Handa, S. S. and Chawla A.S., Fitoterapia, 1989, 60,195. Marles, R.J. and Fransworth, N.R., Econ. Med. Plant Res., 1994, 6, 149. Jayasooriya, A.P., Sakono, M., Yukizaki, C., Kawano, M. and Fukudam, N., J. Ethnopharmacol., 2000, 72, 331. Gurbuz, I., Akyuz, C., Yesilada, E. and Sener, B., J. Ethnopharmacol.,2000, 71, 77. Naseem, M.Z., Patil, S.R. and Patil, R.S., J. Ethnopharmacol., 1998, 61, 9-16. Akhtar, M.S., Athar, M.A. and Yaqub, M., Planta Medica, 1981, 42, 205. Ahmad, N., Hassan, M.R., Halder, H. and Bennor, M.S., Bangladesh Med. Res., 1999, 25, 11. Lotlikar, M.M. and Rajarama, M.R., Ind. J. Pharm., 1966, 28, 129.
Conclusions
The proposed HPTLC method is rapid, simple and accurate for quantitative estimation of charantin in different marketed polyherbal formulations and small fruits, big fruits and fresh fruits variety of M. charantia. The recovery values of charantin were
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Determination of Cefadroxil in Bulk Powder and its Dosage Forms by Spectrophotometric Method
Haresh M. Patel*, Bhanubhai N. Suhagia, Shailesh A. Shah, and Ishwarsinh S. Rathod Abstract: A simple and sensitive spectrophotometric method has been developed for determination of cefadroxil in bulk powder and its pharmaceutical dosage forms. The proposed method was based on reaction of primary amine group of cefadroxil and acetyl acetoneformaldehyde reagent, which gives a yellow coloured chromogen with absorption maxima at 400.0nm. The method is found to be linear in the concentration range of 20 - 100g/ml with regression co-efficient of 0.9998. No significant difference was found between proposed and official method when one and two tailed t-test were applied. Various reaction parameters (reagent concentration and time for maximum colour development, effect of temperature on reaction and effect of pH of reagent) were optimized. The method was validated and can be used successfully to assay cefadroxil in bulk powder and its pharmaceutical dosage forms i.e. tablets and capsules.
Introduction Cephadroxil is first generation of cephalosporin antibiotic which act on gram positive bacteria. Chemically2-3 it is 7 - [(R) - 2- amino 2-(4-hydroxylphenyl) acetamido]-3-methyl-3-cepham-2carboxylic acid monohydrate. It is official in Indian Pharmacoepia, United State Pharmacopoeia2 and British Pharmacopiea.3 Literature survey reveals that cefadroxil is estimated in pharmaceuticals and biological fluids by spectrophotometric,4-5 HPLC,6-9 HPTLC10 and spectroflourimetric methods.11 In the proposed investigation, an attempt has been made to develop a simple, accurate and reproducible spectrophotometric method for estimation of cefadroxil in pharmaceutical formulations. The proposed method is based on reaction of primary amine group of cefadroxil with acetyl acetoneformaldehyde reagent which gives a yellow coloured chromogen with absorption maxima at 400.0nm. The proposed method was successfully applied for determination of cefadroxil in bulk drug and its pharmaceutical formulations.
1 1
formaldehyde solution and glacial acetic acid were purchased from S. D. Fine Chem. Pvt. Ltd., Mumbai and double distilled water were used. Tablets and capsules are dosage forms of cefadroxil, which were procured from local market. Apparatus Double beam Shimadzu 160A UV visible spectrophotometer was employed for spectral measurement. Thermostatically controlled water bath was used to control temperature of reaction mixture. Preparation of reagent solution Weighed accurately sodium acetate (1.64 gm) in a 100 ml volumetric flask, add glacial acetic acid (1.2 ml) and formaldehyde solution (15 ml) and mixed thoroughly. Acetyl acetone (7.8 ml) was added and final volume was adjusted upto 100 ml with distilled water. pH of the buffered solution was adjusted to 3.9. Freshly prepared reagent was used in the study. Preparation of standard solution of cefadroxil Cefadroxil (50 mg) was accurately weighed and transferred to 50 ml volumetric flask. It was dissolved in distilled water (25.0 ml) and diluted to 50 ml with same to obtain the final concentration of 1000 g/ml. Determination of wavelength of maximum absorbance In a 25 ml volumetric flask, standard cefadroxil solution (1.0 ml) and reagent solution (4.0 ml) were pipetted successively and

Reagents Cefadroxil working standard was procured as a gift sample from Torrent Pharmaceuticals Ltd., Ahmedabad. Acetyl acetone (freshly distillated, ExcelaR), anhydrous sodium acetate,
Department of Quality Assurance, L. M. College of Pharmacy, Ahmedabad, Gujarat, India. *For correspondence: patelhary@rediffmail.com
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mixed thoroughly. The reaction flask was allowed to stand at 37OC for 30 min with occasional shaking. Volume was adjusted upto the mark with distilled water. Absorbance of the coloured solution was scanned on Shimadzu UV-visible spectrophotometer from 800 nm to 200 nm against reagent blank. Maximum absorbance was obtained at 400.0nm. The Beers law was obeyed in the range of 20-100 mg/ml. Table 1: Optical characteristics of the proposed method Parameters Wavelength of maxima (nm) Beer's Law limit (g/ml) Molar absoptivity (lit/mole/cm) Sandell's sensitivity (g/ml/cm2/0.001 abs. unit) Regression equation (Y#) Slope (b) Intercept (a) Correlation coefficient (r) Relative standard deviation (%) Recovery (%)
# D
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Values 400 20-100 2.57 103 0.143 10-2 0.0067 0.0006 0.9998
D
Determination of cefadroxil in capsules /tablets

Twenty capsules/tablets were weighed accurately and emptied/powdered it. The powder equivalent to cefadroxil (25 mg) was transferred into test tube containing distilled water (5.0 ml) and vortex it for 2 min. Contents of the test were transferred into 25 ml volumetric flask, residue of the test tube was washed with distilled water (5 ml) and sonicated it for 30 min. The content of flask was transferred into a centrifuge tube and centrifuged it for 10 min at 2000 rpm. The solution was filtered through a Whatman No. 41 filter paper. Residue was washed with distilled water (10 ml). Filtrate and washing were combined in a 25 ml volumetric flask and volume was adjusted up to mark with distilled water. The solution (1.0 ml) was analyzed as above. Amount of cefadroxil was computed, by comparing with the standard simultaneously.
0.96-3.33 99.17-101.10
means Y=a+bC, where C is concentration (g/ml) and Y is absorbance unit. means five replicate samples. + means three replicate samples.
The proposed spectrophotometric method for estimation of cefadroxil is simple, accurate and reproducible, precise and can be employed for routine in-process quality control analysis and quantitative determination of cefadroxil from bulk drug and its pharmaceutical preparations i.e. tablets and capsules.

It is reported that amino beta lactum antibiotics (ampicillin, amoxicillin, cephalexin, cephradine) were determined by spectrophoto-metric method using acetylacetone formaldehyde reagent.12 In proposed method, the principle is based on reaction of acetyl acetoneformaldehyde reagent with primary amine group of cefadroxil which gives a yellow coloured chromogen with absorption maxima at 400.0 nm. The colour is found to be stable for more than 1 h. In the proposed method, various parameters such as reagent concentration, time for maximum colour development, effect of temperature on reaction and effect of pH of reagent were studied and optimized to obtain maximum colour intensity. The optical characteristics of cefadroxil such as Beer's law limit, Sandell's sensitivity, molar extinction coefficient and percentage relative standard deviation of proposed method for determination of cefadroxil were determined (Table 1). The recovery was in the range of 99.17-101.10%. The good recovery confirmed the accuracy and the specificity of the proposed method and the lack of interference from the common excipients, film coating materials and colourant used in the manufacture of tablets and capsules. The developed method was also compared with official method4 and no significant difference was observed.
Acknowledgements
The authors are thankful to Torrent Pharmaceuticals Limited, Ahmedabad for supplying gift sample of cefadroxil.
References
Indian Pharmacopoeia, Vol-I, Published by controller of publication, Delhi, India, 1996, 144. 2. United State Pharmacopoeia, XXIII, NF XVIII, The USP convention, Inc, Rockville, MD, 1995, 197. 3. British Pharmacopoeia, Addendum, Published by HMSO Electronical publication sales, London, UK, 1993, 1305. 4. P.R. Mishra, and S.K. Jain, Indian Drugs, 1998, 35, 600. 5. M.V. Prasad, R. Nagaraju, and T.V. Narayan, Indian J. Pharm. Sci., 2004, 66, 341. 6. C. Hendrix, C. Wijsen, E. Roets, J. Hoogmartens, and Li. Ming. Yun, J. Chromatogr., 1993, 628, 49. 7. C. Hendrix, C. Wijsen, E. Roets, J. Hoogmartens, and Li. Ming. Yun, J. Chromatogr., 1993, 634, 257. 8. Y.M. Shinde, and C.V. Shabadi, Indian Drugs, 1997, 34, 399. 9. Zhi Yuan, Henry Q. Russlie, and Daniel M. Canafax, J. Chomatogr. B Biomed. Appl., 1995, 674, 93. 10. C.V. Shabadi, and A.R. Shelar, Indian Drugs, 1998, 35, 766. 11. Aly Fatma A., Alarfeffj, Nawal A., and Alwarthan A., Talanta, 1998, 47, 471. 12. I.T. Patel, M.B. Devani, and T.M. Patel, J. AOAC Intern., 75, 1992, 994. 1.
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Reverse Phase HPLC Determination of Celecoxib in Dosage Forms

T. E. G. K. Murthy*1, Y. A. Chowdary1, K. Narendra Kumar1, D. Gowri Sankar2 and B. Durvasa Rao2
Abstract: A simple, accurate, precise and reproducible method has been developed and validated for the estimation of celecoxib in dosage forms by RP-HPLC. Chromatography was carried out on an ODS column using a mixture of 0.2% v/v of glacial acetic acid and acetonitrile (32:68 v/v) as the mobile phase at a flow rate of 1ml/min. Valdecoxib is used as an internal standard and the detection was done at 260 nm. The retention times of drug and internal standard were14.177 and 4.50 min. respectively. The method produced linear responses in the concentration range of 2- 20 g/ml of celecoxib. The method was found to be applicable for determination of the drug in capsules as well as in tablets.
Introduction
Celecoxib is selective COX-2 inhibitor and it is chemically 4 (5Methyl phenyl)-3-(trifluromethyl) pyrazole-1-yl) benzene sulfonamide. It has been found to be effective analgesic and antiinflammatory agent for the management of arthritis, post operative dentel pain and low back ache.1 A literature survey reveals the reports of spectrophotometic,2,3 spectrofluorimetric,4 HPLC,5,6 LC,7 MEKC,8 HPTLC9 methods for the determination of celecoxib in pharmaceutical dosage forms and in biological fluids. In this communication we report a new reverse phase HPLC method for the determination of Celecoxib in formulation, which is simple, selective, precise and reproducible.
column maintained at ambient temperature. The detection was carried out at 260 nm. Estimation of celecoxib: About 50 mg of celecoxib was weighed accurately and transferred into a 50 ml volumetric flask and dissolved in 25 ml of mobile phase. The solution was sonicated for 15 min and then the volume made up with a further quantity of the mobile phase to get 1 mg/mL solution. Subsequent dilutions of this solution ranging from 2 to 20 g/ml were made in 10mL volumetric flasks after addition of 1 mL of a 100 g/ml valdeicoxib solution as an internal standard to each solution, 20L of the solution was injected each time into the column. Each of the dilutions was injected five times in to the column and the corresponding chromatograms were obtained. From these chromatograms, the retention times and the areas under the peaks of the drug and internal standard were noted. Using these area values, mean ratio of peak area of the drug to that of internal standard for each dilution were calculated. The regression equation of the drug concentrations over these ratios was computed. This regression equation was later used to estimate the amount of celecoxib in pharmaceutical dosage forms. To check intra-day and inter-day variation of the method, solutions containing 5, 10 and 15 g/ml of celecoxib were subjected to the proposed HPLC analysis. The intraday variation and intraday variation were found to be 1.45 and 1.51 respectively. The drug recovery studies were carried out by adding Known amount of celecoxib to the pre analyzed drug samples and then analyzing them by the proposed HPLC method. Estimation of the drug in Pharmaceutical dosage form: Some formulations were chosen for testing the suitability of the proposed method to estimate celecoxib. For this 20 tablets/capsules were weighed and powered. An accurately
Experimental Work
Chemicals and solvents: HPLC grade methanol (Merck, India), water (triple distilled prepared by all-glass distillation apparatus), and AR grade glacial acetic acid were used for preparing mobile phase. Pure samples of celecoxib and valdecoxib and commercial samples of tablets namely ICEL and capsules namely ZYCEL were employed in the study. Chromatographic conditions: An isocratic HPLC system (Shimadzu) consisting of LC-10 AT liquid pump, SPD-10 AVP UVVisible detector, an ODS C-18 RP column (4.6mmI.D250mm), 20L Hamilton injecting syringe and Spinchrome software was used. Shimadzu AS200 electronic balance was used for weighing the materials. Freshly prepared 32:68 v/v mixture of 0.2% glycial acetic acid and acetonitrile was used as the mobile phase. Methanol, glacial acetic acid and water were filtered through the 0.45 membrane filter and sonicated before use. The flow rate of mobile phase was maintained at 1 ml/min. The
1
Bapatla College of Pharmacy, Bapatla. 2Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam
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weighed portion of this powder equivalent to 50 mg of the celecoxib was transferred to the 50 ml volumetric flask containing 25mL of the mobile phase. The contents of the flask were allowed to stand for 6 hours with intermittent sonication to ensure complete solubility of the drug and then filtered through a 0.45 membrane filter. Approximate volume of this filtrate equivalent to 10 g/ml of drug along with 1mL of valdicoxib solution were taken in a 10mL volumetric flask. The contents of the flask were made up to the volume with mobile phase and mixed well. 20L of the solution was then injected into the column. The mean peak area ratio of the drug to that of the internal standard of five such concentrations was calculated and the drug content in the tablet was quantified using the regression obtained from the pure samples.
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The drug content in the tablet was quantified using the proposed analytical method .The mean amount of celecoxib obtained in different dosage forms is shown in Table1. This reveals that the method is quite precise. High recoveries of above 99.8% of celecoxib from pre analyzed samples of pure drug and its formulations indicate appreciable accuracy of the proposed HPLC method. The absence of additional peaks in the chromatogram indicates non interference of common excipients used in formulations.
Table 1: Assay of Celecoxib in Dosage Forms Formulation Labeled amount Mean amount found Mean labeled (mg) of drug by the the proposed Amount (%) of drug method Tablet (CELEDOL) Capsule (ZYCEL) 100 100 99.6 99.83 99.6 99.83
Results and discussions

The present study was aimed to develop a sensitive, precise and accurate HPLC method for the analysis of celecoxib in pharmaceutical dosage forms. For this 32:68 v/v mixture of 0.2% glacial acetic acid and acetonitrile was found to be most suitable mobile phase as the chromatographic peaks obtained with the system were better defined and resolved and all almost free from tailing. Under the above-mentioned chromatographic conditions, the retention times were obtained for celecoxib and the internal standard were 14.177 and 4.50 min respectively. A model chromatogram is shown in Fig.1.
(mV) 0.5
Conclusion
It can be concluded that the proposed HPLC method is precise, sensitive and reproducible for the analysis of celecoxib in pharmaceutical dosage forms .The method was duly validated by the evaluation of the required parameters.
Acknowledgments
The authors are grateful to Bapatla Education Society, Bapatla for providing the necessary facilities to carry out the research work.
Voltage
0.0 -0.5 -1.0
References
1. 2.
0 5 10 15 20
Physician's Desk Reference, 57th Ed. 2003, 2577. Muralimohanbabu ,G.V.,Gouri Shankar ,V., Narayan Chevuvu, P.S., Hima Shankar, K., Rajendra Kumar, K., Raman Murty, K.V.; Acta Ciencia Indica, 2002, 28(3), 167. Kiran Kumar, M., Nagoji, K.E.V., Kanna Rao, K.V., Bhanoji Rao, M.E.; Acta Ciencia Indica, 2003, 29(1), 11. Patricia, D., Mariela, B., Miguela, C.; Analytical & Bioanalytical chemistry, 2003, 376(7), 1141. Zhonggu, Ke., Wang, H., Zheng, H., Luo, S.; Zhongguo Yiyuan Yaoxue Zazhi ,2003, 23(4), 205. Jaya Sagar, G., Kumar, M.K., Chandrasekhar, K., Prasad, P. S., Rao, Y.M.; Pharmazie, 2002, 57(9), 619. Srinivasu, M.K., Narayana, C.L., Rao, D.S., Reddy, G.O.; J. Pharma. Biomed. Anal., 2000, 22(6), 949. Srinivasu, M.K., Rao, D.S., Reddy, G.O.; J. Pharma. Biomed. Anal., 2002, 28 (3-4), 493. Sane, R.T., Swati, P., Sachin, K.; Journal of Planar Chromatography Modern TLC, 2004, 17(1), 61.
Retention Time (min)
3. 4.
Fig 1. Typical HPLC Chromatogram of Celecoxib
The ratios of the peak area of celecoxib to that of internal standard for different concentrations setup as above were calculated. The peak area of the both the drug and internal standard were reproducible as indicated by low coefficient of variation (1.21). A good linear relationship was observed between the concentration of the celecoxib and the respective ratio of peak areas. The mathematical expression obtained from the regression curve constructed by linear regression fitting was y =4.4862x+0.0685 (where 'y' is the ratio of area under the curve of the drug to that of the internal standard and 'x' is the concentration of celecoxib).
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Simultaneous Estimation of Chlorzoxazone and Tramadol Hydrochloride in Tablet Formulation by Two Wavelength Spectrophotometry
M.P. Puranik*, Anjali Hirudkar, S.J. Wadher, and P.G. Yeole
Abstract: A simple, rapid and economical method for simultaneous estimation of tramadol hydrochloride and chlorzoxazone in tablet formulation has been developed. Chlorzoxazone is quantized using two wavelengths, 266.00 nm and 281.10 nm whereas tramadol hydrochloride is quantized at its lmax, 272.20 nm in 0.1 N methanolic NaOH solution. The Beer's law is obeyed by tramadol hydrochloride and chlorzoxazone in concentration range of 30-300 g/ml and 5-50 g/ml respectively.
Introduction
Chlorzoxazone (CLZ), 5-Chloro-2-benzoxazolinone is official in USP and used as skeletal muscle relaxant. Tramadol hydrochloride2 (TRM), () Trans-2-[(Dimethylaminomethyl]1- (3- methoxyphenyl) cyclohexanol hydrochloride, is official in EP and is used as opoid analgesic. A tablet formulation containing TRM 50 mg and CLZ 250 mg is introduced in the market for the treatment of low back pain and cervical spondylosis. EP describes potentiometric method for assay of TRM and USP describes HPLC method for assay of CLZ. Literature survey revealed that spectrophotometric3, 4 and RPHPLC5, 6, 7 methods are reported for estimation of TRM in pharmaceutical formulation and biological fluids. Spectrophotometric8, and RP-HPLC9 methods are reported for estimation of CLZ in combination with other drugs and. The present work describes development and validation of a simple, precise and accurate two wavelength method for simultaneous estimation of TRM and CLZ in tablet.
1
concentration of 600 g/ml. 2. Chlorzoxazone standard stock solution (0.1 mg/ml) was prepared by transferring accurately 10 mg portion of CLZ in 100 ml volumetric flask and volume was made to mark with 0.1 N methanolic NaOH to give concentration of 100 g/ml. Selection of wavelengths : Selection of analytical wavelengths was done by taking pure samples of TRM and CLZ which were separately dissolved in 0.1 N methanolic NaOH to give two solutions of 60 g/ml and 10 g/ml respectively. They were scanned in the wavelength range of 200 to 400 nm. From the overlain spectra (Figure 1), the wavelengths selected for estimation of CLZ, were 266.00 nm and 281.10 nm, where TRM has same absorbance and estimation of TRM was done at its lmax, 272.20 nm.
Figure 1: Overlain Spectra of TRM and CLZ
2.000 1.500 1.000
272.20(TRM)

Instrument : UV-VIS double beam spectrophotometer, modelShimadzu UV 2401 PC with 1 cm quartz cells. Standard stock solutions 1. Tramadol hydrochloride standard stock solution (0.6 mg/ml) was prepared by transferring accurately weighed 60 mg portion of TRM in 100 ml volumetric flask and volume was made to mark with 0.1 N methanolic NaOH to give
0.500 0.000
200.0 200.0 266.0 281.10 300.0 350.0 400.0
Wavelength (nm.)
Department of Quality Assurance, Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha, (MS) *For correspondence - Email: sjwadher@yahoo.com
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Linearity : Two series of different concentrations in range of 30-300 g/ml for TRM and 5-50 g/ml for CLZ were prepared from stock solutions. The absorbances of TRM dilutions were noted at 272.20 nm and the absorbances of CLZ dilutions were noted at 266.00 nm and 281.10 nm. The difference in absorbance of CLZ at these two wavelengths was calculated and calibration curves were plotted as absorbance versus concentration for TRM and as absorbance difference versus concentration for CLZ. The curves show linearity in the range of 30-300 g/ml for TRM and 5-50 g/ml for CLZ. Standard solution : Standard solution was prepared by mixing aliquot portions of TRM and CLZ standard stock solutions and diluting it with 0.1 N methanolic NaOH to get final concentration of TRM and CLZ, 60 g/ml and 10 g/ml respectively. Sample solution : Twenty tablets of brand MUZOX (Stedman Pharmaceuticals, Label claim CLZ 250 mg and TRM 50 mg) were weighed and finely powdered. Accurately weighed tablet powder equivalent to 100 mg of CLZ was taken into 100 ml volumetric flask. To it 580 mg of pure TRM was added and sonicated for 5 min with 50 ml 0.1 N methanolic NaOH. Volume was made to mark. Aliquot portion was further diluted to achieve final concentration of 60 g/ml for TRM and 10 g/ml for CLZ. Absorbencies of standard and sample solution were noted at 266.00 nm, 281.10 nm and 272.20 nm. The concentrations of two drugs in sample solution were determined. Table 1: Summary of Validation Parameters. 1. 2. 4. 5. PARAMETERS Linearity range (g/ml) Accuracy (%) Precision (R.S.D.)* Ruggedness Intraday(n=3) Interday(n=3) TRM 30-300 g/ml 99.99 0.0038 99.95 100.05
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CLZ 5-50 g/ml 100.50 0.0084 100.82 100.18
R.S.D. is relative standard deviation were found to be linear in concentration range of 30-300 g/ml and 5-50 g/ml for TRM and CLZ respectively.
Conclusion
The results indicated that the proposed two wavelength method was simple, accurate, precise. Hence it can be used for routine analysis which would involve determination of absorbance of sample and standard solutions at the three wavelengths and simple calculations.
References
1. 2. The United States pharmacopoeia 24, US Pharmacopoeial convention, Rockville, 2000, 302-303. European Pharmacopoeia, 5th Edi. European department for the quality of medicines within the council of Europe, Strasbourg, 2005, 2607- 2608. Abdellatef H.E. Kinetic Spectrophotometric Determination of Tramadol Hydrochloride in Pharmaceutical Formulation, J. Pharm. Biomed. Anal. 2002, 29(5), 835-842. Rajput S.J. and Trivedi P.D. Assay of Tramadol Hydrochloride by Spectrophotometry, Indian Drugs, 2000,-38 (2), 100-101. Pederson R.S., Brosen K. and Nielsen F. Enantioselective HPLC Method for Quantitative Determination of Tramadol in Plasma and Urine. Application to Clinical Studies. Chromtographia, 2003, 57 (56), 279- 285. Nobilis M., Kopecky J., Kvetina J., Chladek J., Svoboda Z., Varisek V., Perlik F., Pour M. and Kunes J. High Performance Liquid Chromatographic Determination of Tramadol and Its ODesmethylated Metabolite in Blood Plasma: Application to a Bioequivalence Study in Human, J. Chromatogr. A, 2002, 949 (1-2), 11-22. Gan S.H. and Ismail R. Validation of High Performance Liquid Chromatography Method for Tramadol and O-Desmethyl Tramadol in Human Plasma Using Solid Phase Extraction. J. Chromatogr. B. 2001, 759 (2), 325-335. Kale U.N., Naidu K.R. and Shingare M.S. Simultaneous Spectrophotometric Determination of Chlorzoxazone and Nimesulide from Combined Dosage Form. Indian J. Pharm. Sci. 2002, 64 (2), 168-169. Salama F.M. High Performance Liquid Chromatography and Second Derivative Spectrophotometric Determination of Chlorzoxazone and Paracetamol in Single and or Combined Dosage Forms. J. Pharm. Sci. 1998, 22, 48-60.
3.
Result
The results obtained by proposed method were in good agreement with the label claim. The additives and excipients did not interfere in analysis of the tablet by proposed method. The method was validated in terms of accuracy, precision, ruggedness, linearity and range. The accuracy of the proposed method was determined by recovery studies. The summary of validation parameters is shown in Table 1.
4. 5.
6.
Discussion
The present study was carried out to develop a simple, rapid, sensitive, accurate, precise and rugged spectrophotometric method for the simultaneous estimation of TRM and CLZ in tablet formulation. TRM shows little absorbance at lower concentrations due to lack of strongly absorbing chromospheric group in its structure7. Hence the ratio of TRM and CLZ was maintained 6:1 by standard addition throughout the analysis, though their ratio in tablet is 1:5, to get the good overlain spectra. The standard deviation values were satisfactorily low and recovery was closed to 100 % indicating the reproducibility, accuracy and precision of the proposed method. The linearity study performed for both the components and the absorbencies
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Spectrophotometric Methods for Quantitative Estimation of Venlafaxine Hydrochloride from Tablet Formulation
S. Pillai and I. Singhvi*
Abstract: Two simple and sensitive visible spectrophotometric methods have been developed for the quantitative estimation of Venlafaxine hydrochloride from its tablet formulation. The developed methods are based on formation of chloroform extractable coloured complex of drug with orange-G and chloro phenol red. The extracted coloured complex of drug with orange-G showed absorbance maxima at 483 nm and linearity was observed in the concentration range of 5- 30 g/ml (method I) and with chloro phenol red showed absorbance maxima at 410 nm and linearity was observed in the concentration range of 5-30 g/ml (method II). Results of analysis for both the developed methods were validated statistically and by recovery studies.
Introduction
Venlafaxine hydrochloride, chemically 1-[2-(dimethyl amino)-1(4-methoxy phenyl) ethyl] cyclohexanol, is recently developed antidepressant drug1. Few analytical methods for estimation of Venlafaxine hydrochloride from biological fluid including HPLC2-4 , capillary electrophoresis5 and HPLC-MS6 are reported.
Experimental Instrument and reagent

A systronics UV/Visible double beam spectrophotometer (model 2101) with 1cm matched quartz cells was used for spectral analysis. All the chemicals used were of analytical grade. The orange-G dye solution (0.3%w/v) was prepared in phosphate buffer pH 4 (method I) and chloro phenol red dye solution (0.3%w/v) was prepared in potassium chloride hydrochloric acid buffer pH2 (method II) and extracted several times with chloroform so as to remove chloroform soluble impurities. Double distilled water was used for the preparation of buffer and standard stock solution of the drug. The tablet samples of Venlafaxine hydrochloride were procured from the local market.
to give several dilutions in concentration range of 5-30 g/ml of drug. To 5 ml of each dilution taken in a separating funnel, 5 ml of orange-G dye solution was added, shaken and allowed to stand for 10 minutes for the formation of coloured complex. The coloured complex was extracted with 5, 3 and 2 ml portions of chloroform and absorbance of the combined chloroform layer was measured at 483 nm against a reagent blank. A calibration curve was prepared by plotting concentration versus absorbance. For method II, in a series of 10 ml volumetric flasks aliquots of standard drug solution of Venlafaxine hydrochloride (100g/mL) in distilled water were transferred and diluted with the same so as to give several dilutions in concentration range of 5-30 g/ml of drug. To 5 ml of each dilution taken in a separating funnel, 5 ml of chloro phenol red dye solution was added, shaken and allowed to stand for 10 minutes for the formation of coloured complex. The coloured complex was extracted with 5, 3 and 2 ml portions of chloroform and absorbance of the combined chloroform layer was measured at 410 nm against a reagent blank. A calibration curve was prepared by plotting concentration versus absorbance.
Preparation of calibration curve

For method I, in a series of 10 ml volumetric flasks aliquots of standard drug solution of Venlafaxine hydrochloride (100g/ml) in distilled water were transferred and diluted with the same so as
Analysis of tablet formulation

For analysis of formulation, twenty tablets of Venlafaxine hydrochloride were accurately weighed and average weight per tablet was determined. The tablets were powdered and powder
Department of Pharmaceutical Sciences, M.L. Sukhadia University, Udaipur E-mail - INDRAJEET_S@yahoo.com, sujitpillai2001@yahoo.com
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equivalent to 20 mg of drug was accurately weighed and was extracted four times with 20 ml portions of distilled water, the combined extract was filtered through Whatman filter paper No.41 into 100 ml volumetric flask. The residue was washed with distilled water and the washings were added to the filtrate, final volume of filtrate was made up to the mark with distilled water. For method I, 0.5 ml of filtrate was diluted to 10 ml with distilled water. This was treated as per the respective procedure for the calibration curve and amount of drug present in sample was computed from respective calibration curve. For method II, 0.5 ml of the filtrate was diluted to 10 ml with distilled water. This was treated as per the respective procedure for the calibration curve and amount of drug present in sample was computed from respective calibration curve. The developed methods were repeated five times for two different strengths of tablet formulations, Results of analysis are reported in Table 1.
Table 1: Analysis of Tablet Formulation Method Formulation Label % of label % Standard Claim Claim Recovery** Deviation (mg/tab) estimated* I II Tablet 1 Tablet 2 Tablet 1 Tablet 2 25 75 25 75 99.80 99.97 99.66 99.30 99.81 99.92 99.33 99.18 0.360 0.253 0.529 0.560
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The proposed spectrophotometric methods for determination of Venlafaxine hydrochloride from tablet formulations are based on formation of chloroform extractable coloured complex of drug with orange-G and chloro phenol red. The pH required for visible spectrophotometric methods was optimized at pH 4 for method I and at pH 2 for method II. The result of analysis for both the developed methods were close to 100% and standard deviation was satisfactorily low indicating accuracy and reproducibility of the methods. Recovery studies were satisfactory which shows that there is no interference of excipient. The developed methods were found to be simple, rapid, accurate and can be used for routine analysis of drug from tablet formulation.
Acknowledgment
The authors are thankful to Dr. Reddy's laboratories, Hyderabad for providing gift sample of Venlafaxine hydrochloride for the present investigation.
References
1. 2. 3. 4. 5. 6. Budvari, S., Eds., In; The Merck Index, 12th Edn., Merck and Co., Inc., White house station, NJ, 1996,1695. Hicks, D.R., Wolaniuk, D., Russel, A., Cavanaugh, N. and Kraml, M., The Drug Monit, 1994, 16, 100. Vu, R.L., Helmeste, D., Albers, L. and Resit , C., J. Chromatogr B. Biomed Sci Appl, 1997, 760, 195. Matoga, M., Pehoureq, F., Titer, K., Dumora, F. and Jarry, C., J. Chromatogr B. Biomed Sci Appl, 2001, 760, 213. Rudaz, S., Stella, C., Balantgorgia, A.E., Fanali, S. and Veuthey, J.L., J. Pharm. Biomed Anal, 2000, 23, 107. Jaran, H., Zhiling, Z. and Huande, L., J. Chromatogr B. Anal. Technol. Biomed Life Sciences, 2005, 33, 820.
* Average of five determinations. **Average of determination at three different concentration levels.
Recovery studies
Recovery studies were carried out for both the developed methods by addition of known quantity of pure drug sample to pre analyzed tablet sample solution at three different levels. The result of recovery studies are reported in Table 1.
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New Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Ketoprofen in Tablet Dosage Form
R.K. Maheshwari
Abstract: A new, simple, environmentally friendly, cost-effective, safe and sensitive spectrophotometric method in ultraviolet region has been developed using 4.0 M Sodium acetate hydrotropic solution as solubilizing agent for the determination of ketoprofen, a poorly water-soluble drug in tablet dosage form. Ketoprofen shows maximum absorbance at 260 nm. Beer's law was obeyed in the concentration range of 4-20 mcg/ml. Results of the analysis were validated statistically and by recovery studies.
Introduction
In hydrotropic solubilization phenomenon, addition of large amount of a solute results in an increase in the aqueous solubility of another solute. Concentrated aqueous solutions of a large number of hydrotropic agents have been employed to enhance the aqueous solubility of many poorly water-soluble drugs.1-15 Sodium salicylate, sodium benzoate, nicotinamide, urea, sodium citrate and sodium acetate are most common examples of hydrotropic agents. Maheshwari1 has analyzed a poorly watersoluble drug, cefixime, in the tablets by spectrophotometric analysis (lmax 330 nm) using three different hydrotropic solutions of urea (8.0 M), Sodium acetate (4.0 M) and Sodium citrate (1.25 M) as solubilizing agents to extract out the drug from fine powder of tablets. Maheshwari2 analyzed frusemide (a poorly watersoluble drug) in bulk drug and in marketed tablets by conducting titrimetric analysis using 2.0 M sodium benzoate solution as hydrotropic solubilizing agent. Maheshwari et al.3 used 2.0 M Sodium benzoate solution to solubilize ofloxacin (a poorly watersoluble drug) from fine powder of its tablets to carryout its spectrophotometric analysis (lmax 332 nm). Maheshwari4 analyzed bulk drug sample of ketoprofen and salicylic acid by titrimetric analysis. Three different hydrotropic solutions of Sodium benzoate (2.0 M), Sodium salicylate (2.0 M) and Sodium acetate (2.0 M) were employed to solubilize ketoprofen to carryout titrimetric analysis. Hydrotropic solutions of Sodium benzoate (2.0 M), urea (8.0 M) and Sodium citrate (1.25 M) were employed to solubilize salicylic acid to carryout titrimetric analysis. Maheshwari5 has analyzed tinidazole in its tablets by
spectrophotometric analysis (lmax 318 nm) using three different hydrotropic solutions of Sodium citrate (1.25 M), Sodium acetate (4.0 M) and urea (8.0 M). Ketoprofen [2- (3 benzoylphenyl) propionic acid] is a widely used non-steroidal anti-inflammatory drug. There was tremendous increase in the solubility of ketoprofen in 4.0 M Sodium acetate (hydrotropic) solution. It was thought worthwhile to employ this solution to extract out ketoprofen (a poorly water-soluble drug) from fine powder of its tablets for spectrophotometric analysis. Presence of sodium acetate did not interfere in the estimation.
Experimental
Instrument : A Shimadzu UV Visible recording spectrophotometer (Model - UV 160 A) with 1 cm matched silica cells was used for spectrophotometric analysis. Materials: Ketoprofen tablets of different batches and ketoprofen drug were supplied as gift samples by Ranbaxy Lab. Ltd., Dewas (India). All chemicals used were of analytical grade.
Methods
Calibration curve : The standard solution (200 mcg/ml) of ketoprofen was made in distilled water. The standard solution was further diluted with distilled water to obtain various dilutions (4, 8, 12, 16 and 20 mcg/ml). A linear relationship was observed over the range of 0 to 20 mcg/ml for ketoprofen (lmax 260 nm).
Preliminary solubility studies of drug

Solubility of ketoprofen was determined in distilled water and 4.0 M Sodium acetate solution at 27 10C. Enhancement in the
Department of Pharmacy, Sri G.S. Institute of Technology and Science, 23, Park Road, Indore E-mail: rkrkmaheshwari@indiatimes.com, Phone 0731-2542213
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solubility of ketoprofen in 4.0 M Sodium acetate solution was Table 2 : Recovery study for spiked concentration of ketoprofen added to the preanalyzed dosage form more than 120 fold (as compared to its solubility in distilled water). Tablet Amount Pure Percent recovery % coeff. Standard Analysis of tablet formulations of ketoprofen by the formulation of drug ketoprofen estimated* of error proposed method: Three different batches of ketoprofen (mg) added (mg) (meanS.D.) variation tablets were used in the present investigation. Twenty tablets I 100 10 98.770.621 0.629 0.358 of ketoprofen (formulation-I) were weighed and ground to a 100 15 98.140-944 0.962 0.545 fine powder. An accurately weighed powder sample 100 20 98.660.818 0.829 0.472 equivalent to 100 mg of ketoprofen was transferred to a 50 ml II 100 10 101.341.022 1.008 0.590 volumetric flask containing 20 ml of 4.0 M Sodium acetate 100 15 100.391.103 1.099 0.637 solution. The flask was shaken for about 5 min to solubilize 100 20 99.810.916 0.918 0.529 the drug and the volume was made upto the mark with III 100 10 101.831.331 1.307 0.768 distilled water. The solution was filtered through Whatman 100 15 100.550.887 0.882 0.512 filter paper # 41. The filtrate was divided into two Parts A and 100 20 98.340.773 0.786 0.446 B. Part A was kept at room temperature for 48 hours to check its chemical stability and precipitation, if any. Part B was * Average of three determinations. diluted sufficiently with distilled water and was analyzed on more than 120 fold as compared to solubility in distilled water. UV-spectrophotometer against reagent blank. Drug content of Therefore, this solution was employed to extract out the poorly tablet formulation was then calculated (Table 1). After 48 hours, water-soluble drug, ketoprofen from fine powder of its tablet Part A solution was analyzed in the same may as Part B solution. formulation. It is evident form Table 1 that percent label claims Tablet formulation-II and III were treated in the same way. were 99.32 0.871, 100.73 1.213 and 101.05 0.938. Percent label Table 1 : Results of analysis of commercial tablet claims are very close to 100 showing the accuracy of the formulations with statistical evaluation proposed methods. Low values of standard deviation, percent Tablet Label Percent label claim Percent coeff. Standard coefficient of variation and standard error validated the proposed method. formulation claim (mg) estimated* of variation error (meanS.D.) Accuracy, reproducibility and precision of the proposed method I 100 99.320.871 0.876 0.503 was further confirmed by percent recovery values. As evident II 100 100.731.213 1.204 0.700 from Table 2, percent recovery values ranged between 98.14 II 100 101.050.938 0.928 0.541 0.944 and 101.83 1.331. Percent recovery values were close to 100 with low values of standard deviation, percent coefficient of * Average of three determinations. variation and standard error. These results validated the Recovery studies proposed method. In order to carryout recovery studies 10, 15 and 20 mg ketoprofen pure drug was added to tablet powder equivalent to 100 mg ketoprofen. In all three cases, 4.0 M Sodium acetate solution was employed for solubilization of drug. After making up the volume to 50 ml in the volumetric flask with distilled water, further dilutions were done with distilled water (after filtering through Whatman filter paper # 41). Appropriate dilutions were analyzed on UVspectrophotometer against reagent blank. Percent recoveries were determined and noted in Table 2. The drug contents of extracts of drug in hydrotropic solution remained same during 48 hours and there was no precipitation of drug within 48 hours. Therefore drug can be analyzed in extracts within 48 hours at least without detrimental effect on drug stability.
Conclusion
Methanol, ethanol, acetonitrile, hexane, diethyl ether, cyclohexane, chloroform, carbon tetrachloride, toluene and acetone have been employed for solubilization of poorly watersoluble drugs for their spectrophotometric analysis. Most of the organic solvents are toxic, costlier and cause pollution. Volatility of such solvents is the cause of inaccuracy of results. Using poorly water-soluble drug, ketoprofen as a model drug, author
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Results of solubility studies of ketoprofen revealed that enhancement in solubility in 4.0 M Sodium acetate solution was
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wants to emphasize on the use of hydrotropic solutions as solubilizing agents. Sodium acetate did not interfere in the spectrophotometric estimation of drug. Thus, other poorly watersoluble drugs can be tested for their solubility in 4.0 M sodium acetate solution. If they have good solubilities in this hydroptropic solution, they can be easily estimated by use of this hydrotropic agent precluding the use of organic solvents. It is thus concluded that the proposed method is new, simple, cost-effective, accurate, safe, environmentally friendly and precise and can be successfully employed in the routine analysis of ketoprofen tablets.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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References
1.
2. 3. Maheshwari, R.K., Indian Pharmacist, 2005, Vol. IV (No. 36), 63. Maheshwari, R.K., Indian Pharmacist, 2005, Vol. IV (No. 34), 55. Maheshwari, R.K., Chaturvedi, S.C. and Jain, N.K., Indian Drugs, (In Press).
Maheshwari, R.K., Asian J. Chem., (In Press). Maheshwari, R.K., Asian J. Chem., (In Press). Jain, N.K., Agrawal, R.K. and Singhai, A.K., Pharmazie, 1990, 45, 221. Jain, N.K. and Patel, V.V., Eastern Pharmacist, 1986, 29, 51. Hussain, M.A., Diluccio, R.L. and Maurin, M.B., J. Pharm. Sci., 1993, 82, 77. Frost, D.V., J. Am. Chem. Soc., 1947, 69, 1064. Rasool, A.A., Hussain, A.A. and Dittert, L.W., J. Pharm. Sci., 1991, 80, 387. Etman, M.A. and Hada, A.H., Acta Pharm., 1999, 49, 291. Agrawal, S., Pancholi, S.S., Jain, N.K. and Agrawal, G.P., Int. J. Pharm., 2004, 274, 149. Ueda, S., Chem. Pharm. Bull., 1966, 14, 2. Saleh, A.M. and Daabis, N.A., Pharmazie, 1974, 29, 525. Poochikian, G.D. and Cradock, J.C., J. Pharm. Sci., 1979, 68, 728.
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Spectrophotometric Method for the Estimation of Valdecoxib and Paracetamol from Combined Dosage Form
K. E. V. Nagoji*, V. Kiran Kumar, K. Ravi Sankar, V. Venkata Rao, Prafulla Kumar Sahu and M. E. B. Rao
Abstract: A simple, accurate and economical spectrophotometric method was developed for simultaneous determination of valdecoxib and paracetamol in combined pharmaceutical dosage forms. Valdecoxib and paracetamol has lmax at 237 nm and 248 nm respectively in methanol, showing linearity in the concentration range of 0.025-1 g/ml and 0.625-25 g/ml respectively. The results of analysis have been validated statistically and recovery studies confirmed the accuracy of the proposed method.
Introduction
Valdecoxib is a non-steroidal anti-inflammatory drug (NSAID) and chemically it is 4-(5-methyl-3-phenyl-4-isoxazolyl) benzene sulfonamide. Paracetamol is an analgesic and anti-pyretic drug. Chemically, it is 4-hydroxy acetanilide and it is official in IP,1 BP2 and USP.3 A review of literature reveals that there are spectrophotometric, HPLC4-9 methods for the determination of valdecoxib and paracetamol individually. But there is no evidence in literature for simultaneous determination of these drugs in combined dosage forms. Hence, in the present investigation, a simple, rapid and reproducible method was developed for the simultaneous determination of these drugs in combined dosage forms.
concentrations of 0.025-25 g/ml and 0.625-625 g/ml respectively. The absorbance was measured at 237 nm for valdecoxib and 248 nm for paracetamol against methanol as blank. Both these drugs obeyed linearity individually and in mixture within the concentration range of 0.025-1 g/ml for valdecoxib and 0.625-25 g/ml for paracetamol. Six replicates of six mixed standard solutions were prepared from standard stock solutions of the two drugs as given in Table 1. The absorbances of the solutions were measured at 237 nm and 248 nm for valdecoxib and paracetamol respectively. Quantitative determination of these drugs was carried from the standard graphs of valdecoxib and paracetamol. Table 1: Concentrations of valdecoxib and paracetamol in mixed standard solutions Concentration (g/ml) Valdecoxib 0.025 0.050 0.100 0.250 0.500 1.000 Paracetamol 0.625 1.250 2.500 6.250 12.50 25.00 Absorbance At 237 nm 0.051 0.108 0.199 0.518 1.020 2.046 At 248 nm 0.062 0.128 0.248 0.611 1.210 2.432
Materials And Methods

An Elico SL-159 UV/Visible spectrophotometer with 1 cm matched quartz cells was used for all absorbance measurements. Methanol (analytical grade) and double distilled water were used for preparing samples. An Afcoset-electronic balance was used for weighing the samples.
Procedure for calibration curve

Standard stock solutions were prepared by dissolving 25 mg of each drug in 25 ml of methanol to get a concentration of 1 mg/ml. From this suitable dilutions were made with methanol to get working standard solutions of 100 g/ml and 10 g/ml respectively. To construct beer's plot for valdecoxib and paracetamol, different aliquots of working standard solutions were taken and diluted with methanol to 10 ml to prepare
Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa-760010
Analysis of commercial formulations

Twenty tablets were weighed and powdered. An accurately weighed quantity of powder equivalent to 25 mg of paracetamol
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was transferred into 25ml volumetric flask and dissolved in methanol. Then the solution was filtered and further diluted with methanol. The absorbances were measured at 237 nm and 248 nm for valdecoxib and paracetamol respectively and the quantity was determined from the standard graphs and the results were as given in Table 2. Table 2: Results of Analysis of Commercial Formulations
Formulation Ingredient Label claim Amount found % label (mg/tablet) (mg/tablet) claimed 20.0 500.0 20.0 495.0 100 99
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sensitivity of the proposed method was ascertained by Sandell's sensitivity and Beer's law range was also calculated and given in Table 4. The results reveal the suitability of the proposed method for the simultaneous determination of valdecoxib and paracetamol. Table 4: Validation And Sensitivity Parameters Of The Proposed Method Parameter lmax (nm) Beer's range (g/ml) Sandell's sensitivity (g/cm2/0.001 AU) Regression equation Slope Intercept Correlation coefficient % RSD Valdecoxib 237 0.025-1 0.0004 Paracetamol 248 0.625-25 0.01303
Tablet 1 Valdecoxib (Valeron Plus) Paracetamol

The proposed method for simultaneous determination of valdecoxib and paracetamol in combined dosage forms was found to be simple, accurate, economical and rapid. Validation of the proposed method was carried out by performing recovery experiments, in which pre-analyzed samples were taken and standard drug was added at 3 different concentration levels. The results were as shown in Table 3. Table 3: Results Of Recovery Studies Concentration of drug added to final dilution (g/ml) Valdecoxib 0.25 0.5 1.0 Paracetamol 6.25 12.5 25 Valdecoxib 97.5 100.0 96.6 Paracetamol 100.0 104.0 105.0 % of recovery
2.044 0.0008 0.999 2.398
0.097 0.0027 0.999 3.050
References
1. Indian Pharmacopoeia, 1996, 554. 2. British Pharmacopoeia, 1998, 994. 3. United States Pharmacopoeia XXIII, 785. 4. Thomas, R., Roets, E. and Hoogmartens, J.; J. Pharm. Sci., 1984, 73, 1830. 5. Erram, S. V., Tipnis, H. P.; Indian Drugs, 1993, 30(3), 116-119. 6. Ravi Sankar, S., Vasudevan, M., Nanjan, M. J., Bijukarian Suresh; Indian Drugs, 1997, 34(1), 663-665. 7. Aloba, OT., Adusumalli, P. S., Nigalaye, A. G., J. Pharm. Biomed. Anal., 1991, 9(4), 335-340. 8. Zhang, JY., Fast, DM., Breau, AP.; J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2003, 785(1), 123-134. 9. Rama Krishna, NV.; J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2004, 802(2), 271.
The validation parameters like percentage RSD, regression equation, slope, intercept and correlation coefficient were calculated and the results were as shown in Table 4. The
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Simultaneous estimation of Pantoprazole and Domperidone in Pharmaceuticals Dosage Forms by RP-HPLC

Singh R. A. *, Arora Saurabh, Kumar Robin, Kumar Dinesh & Agarwal A.K Abstrict : A simple, precise, rapid and reproducible method has been developed and validated for the simultaneous estimation of pantoprazole and domperidone in pharmaceuticals tablet dosage forms by RP-HPLC. The separation was achieved using RPC-18 end capped (5m) 250 x 4.0mm column in isocratic mode with mobile phase Buffer: Methanol (25:75v/v) at a flow rate of 1.0ml/min, detection was carried out at 285nm using a UV detector. The retention time for pantoprazole was 2.96 and for domperidone was 5.4 mints.The percentage recovery for pantoprazole and domperidone was found to be 99.0 to 101.0% w / w.
Introduction
Pantoprazole is a proton pump inhibitor and used for the treatment of peptic ulcer disease or gastro oesophageal reflex disease.1 It is not official in any pharmacopoeia. Domperidone is a gastro kinetic and antiemetic drug. It is a peripheral dopamine 2receptor antagonist.2 It is official in B.P.3 The combination of both these drugs are available in the market as tablet dosage forms. The literature survey revealed that there are number of methods have been reported for the estimation of Pantoprazole and Domperidone individually or in combination with other drugs.4,5,6 However there is one method7 reported for the simultaneous estimation of these drugs in combined dosage forms. The present method was developed keeping the current regulatory requirements in mind. This paper presents a simple accurate reproducible and rapid method for the simultaneous estimation of both drugs in combined dosage forms.
Mobile Phase: 250ml of buffer and 750ml of methanol were mixed and filtered through 0.45m fitter paper. Buffer: - 2.54 di -Sodium Phosphate and 2.72 Potassium di-hydrogen phosphate were dissolved in 1000ml water.
Standard Stock Solution

a) Accurately weighed 40mg of Pantoprazole Working standard was dissolved in 20ml of mobile phase in 50ml volumetric flask and diluted to the mark with mobile phase (0.8 mg / ml-1). Accurately weighed 50mg of Domperidone Working Standard was dissolved in 20ml of Dimethyl Formamide in 50ml volumetric flask. Make the volume with Dimethyl Formamide and filter through 0.45 m filter paper. (1mg/ml-1).
b)
Mixed Working Standard

Transfer 2ml of Standard stock solution A and 10ml of Standard Stock Solution B in a 100ml volumetric Flask, mix and makeup volume with mobile phase.
A Jasco Isocratic Liquid Chromatographic system, equipped with model PU-980 pump & Rheodyne injector with 20ml fixed loop and PU-975 UV-Detector.
Sample Preparation
20 Tablets were weighed and crushed to fine powder. A powder equivalent to 40mg of Pantoprazole was weighed accurately and dissolved in 10ml of Dimethyl Formamide and volume was made up to 50ml with mobile phase the solution was filtered through 0.45 m filter paper. Dilute 5ml of the filtrate to 50ml with mobile phase.
Chemicals & Reagents

Working Standard of Pantoprazole and Domperidone. Acetonitrile and Water of HPLC Grade and dimethyl formamide, di -Sodium Phosphate and potassium di-hydrogen phosphate of AR Grade.
* For Correspondence Dr. R.A. Singh 4/9, Kirti Nagar Indl. Area, New Delhi - 15 Phone: 25457922, 25457923
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Chromatographic Conditions Mode of separation Column Reversed Phase Isocratic RPC-18ec (5m) column 250 x 4.0mm pre-washed with methanol (15 minutes) & equilibrated with mobile phase (30 minutes) prior to analysis. 250ml of buffer and 750ml of methanol were mixed and filtered through 0.45m fitter paper. 1.0ml/min U.V. 285nm 1.0
Intensity (AU)
P TR
Domperidone and 0.064mg.ml - 1 to 0.096mg.ml - 1 for Pantoprazole. The chromatogram was scanned at 285nm, being the absorption maxima of Domperidone, the minor component in the formulations. A simple and accurate reversed phase HPLC method has seen proposed for simultaneous estimation of Pantoprazole and Domperidone in combined dosage form. The assay value and recovery data indicate that the method is free from interference of excipients and can be effectively used for routine quality control.
2.96, PANTOPRAZOLE
Mobile phase
Flow rate Detector wave length Chart speed Temperature
0.8 0.6 0.4 0.2 0.0 0 2
Ambient
Procedure
The column was flushed with mobile phase till stable base line obtained, 20l of standard and sample dilution were injected separately under the chromatographic condition described above and scans were recorded each solution was run 5 times. The amount of Pentaprazole & Domperidone in sample was calculated by comparing the peak area ratio from standard.
5.40, DOMPERIDONE
10
12
14
Retention Time (Min.) Fig. 1: Typical HPLC chromatogram of sample containing Pantoprazole and Domperidone.
Recovery
To establish the accuracy, reproducibility and precision of the proposed method, recovery experiments were carried out by spiking the pre-analysed sample at three different levels. The results are recorded on Table 1. Table 1: Simultaneous Assay of Pantoprazole and Domperidone in Dosage Formulation. S.N. Claim (mg) 40.0 40.0 Pantoprazole Found (mg) 39.6 39.5 Recovery (%) 99.0 98.75 Domeperidone Claim (mg) 10.0 10.0 Found (mg) 9.94 9.95 Recovery (%) 99.4 99.5
References
1. 2. 3. 4. Extra Pharmacopoeia Martindale, 32nd edition, 1207.3. Current Index of medical specialties, 22(3), July-sep 1999. British Pharmacopoeia, Published by Her Majesty Stationary Office, 2003, London Argekar A.P., Shah S.J., Simultaneous determination of cinnarizine and Domperidone maleate from tablet dosage by reverse phase ion pair high performance liquid chromatography, J. Pharm. Biomed. Anal., May 1999,19(6), 813-817. Zavitasanos, A.P., Mac Donald C., Bassoo E., Gopaul D., Determination of Domperidone in human plasma and human breast milk by high-performance liquid chromatography-electrospray mass spectrometry, J. Chromatogr. B. Biomed. Appl., 25 Jun1999, 730 (1), 9-24. Yamamoto K., Hagino M., Kotaki H., Iga I., Quantitative determination of Domperidone in rat plasma by high detection, J. Chromatogr B. Biomed. Appl., 11 Dec 1998, 720 (1-2), 251-255. Manoj, K and Abbazhagan S. Reverse phase high performance liquid chromatographic method for simultaneous Estimation of Domperidone And Pantoprazole from tablet formulation. Indian Drugs 41(10), 604-608,2004.
5.
1. 2.
*
Average of three determinations. After spiking the pre-analysed sample at different levels.
6.
**
Results & Discussion

Under proposed chromatographic conditions, both the drugs, Pantoprazole and Domperidone were well separated (Fig-1) with retention time of 2.96 and 5.4 min respectively and the linearity was found in the range of 0.016mg.ml-1 to 0.024mg.ml-1 for
7.

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H.P.L.C Method for Simultaneous Estimation of Tannic acid, Gallic acid, Chebulinic acid and Ethyl gallate in Terminalia chebula fruits
K. Jayaram Kumar, S.P. Bhatnagar
Abstract: Terminalia chebula has been mentioned in traditional system of medicine for many diseases. This paper presents a scientific method for establishing its uses based on composition of various chemical constituents. H.P.L.C method has been developed for simultaneous estimation of tannic acid, gallic acid, chebulinic acid and ethyl gallate in different samples of Terminalia chebula. The results have shown a considerable change in these phyto nutrients in different samples of fruits of Terminalia chebula, which gives a new direction into scientific basis of its traditional uses.
Introduction
Accuracy in recording or observing the medicinal use of a plant, determining whether the ethno medical use can be demonstrated under scientific conditions in laboratory, and/or chemical characterization of compounds and role of placebo effect are important issues that need to be verified in the development of drugs of plant origin. Terminalia chebula is a moderate size to large deciduous tree attaining a height of 15-24 m with leaves ovate, elliptic or obovate, glabrous to villous beneath. Flowers are yellowish white, in axillary and terminal often-paniculate spikes. Fruits are glabrous, shiny, ellipsoidal, obovoid or ovoid drupes, yellow to orange brown in color hard when ripe, stone very thick, bony and rough. Earlier studies on Terminalia chebula have II (Singh et al., 1990 and Kundu et al., 1992) galloyl esters of glucose, corilagin chebulinic, chebulagic acid and many other important constituents.
(10:5:85). The flow rate of the eluent was 1.2 ml/min. The oven o temperature had been kept at 40 C. Purging had been done for 2.0 min. with solvent system after each run. The wavelength used for detection is 264 nm. tannic acid, gallic acid, chebulinic acid and ethyl gallate were eluted at 25.976, 3.410, 27.755 and 3.787 minutes respectively.
Calibration plot for tannic acid, gallic acid, chebulinic acid and ethyl gallate
Different concentrations (range: 5 mg/ml1ml/mg) each of tannic acid, gallic acid, chebulinic acid and ethyl gallate had been prepared in methanol separately and repeated injections (20 m1 n=5) of each concentration had been subjected to H.P.L.C and the respective peak area had been noted down in Table 1. Calibration plots have been drawn as in Figure 1. by incorporating the amount injected vs their corresponding peak areas in each case (i.e. for tannic acid, gallic acid, chebulinic acid and ethyl gallate). Regression equations have been calculated as y=13.1067x+3.39668 for tannic acid, y=32.6678x+0.2128 for gallic acid, y=8.7839+1.26007 for chebulinic acid and y=22.8258x+2.0464 for ethyl gallate.
Material and methods

Samples of fruits of Terminalia chebula Retz were obtained from different regions of India and Nepal. Each sample was given specific code for identification purposes. The fruit were properly dried before analysis and stored in airtight container as used in the normal process of handling.
Optimization of extraction procedure:

Four samples each of 100.0 g accurately weighed T. chebula fruit powder were refluxed with methanol (50 ml) for 30, 60, 90, 120 minutes respectively. The resulting solutions were filtered concentrated under vacuum redissolved in methanol (15 ml) and their final volumes were made upto 25.0 ml. Aliquots (20 mL) of each sample were subjected to H.P.L.C. The marc obtained from
H.P.L.C assay of tannic acid, gallic acid, chebulinic acid and ethyl gallate
An inertsil ODS-3V had been used. The mobile phase consisted of Acetonitrile: Methanol: Phosphate buffer (40 mM, pH 3.0)
Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra

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Table 1: Calibration plot data for tannic acid, gallic acid, chebulinic acid and ethyl gallate
Amount Tannic acid Gallic acid Chebulinic acid Ethyl gallate injected Mean peak CV Mean peak CV Mean peak Cv Mean peak CV (mg/) area area area area 0.25 0.5. 0.75 1.0 2.0 4.0 8.0 16.0 6.59 13.21 26.63 43.12 85.89 171.12 5.1 2.1 0.3 2.7 2.6 5.535 11.021 21.873 42.943 65.012 1.1 2.5 2.7 1.1 0.5 1.123 2.193 4.201 8.503 2.1 0.5 0.6 1.1 4.984 5.813 11.583 22.943 45.613 90.845 181.52 362.14 0.9 4.1 3.2 0.7 2.1 3.4 2.9 2.3
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Table 2: Contents of tannic acid, gallic acid, chebulinic acid and ethyl gallate in different cycles of extraction
Reflux % (w/w) of constituents Mean S.D period Tannic acid min
30 60 Gallic acid Chebulinic acid Ethyl gallate 0.01420.00423
0.8740.0026 0.11320.00125 0.1820.00326
0.8920.00123 0.11480.00145 0.01910.000286 0.01440.00125
90 X 1 0.9280.00235 0.10560.00164 0.01940.000243 0.01470.00184 90 X 2 0.0560.00117 0.00460.00086 0.00950.000125 0.00110.00094 90 X 3 0.0240.00065 0.00240.00043 NDf 90 X 4 NDf 120 NDf NDf 0.9340.00624 0.11490.00156 0.01960.0045 NDf Ndf
17.024 2.4 34.215 0.2 69.013 2.5 139.126 3.1
0.9 132.164 3.4 521.43 1.2
342.013 0.7 263.431 0.6 685.123 1.5
0.01480.00156
Ndf = not detected phytonutrients. This variation is one of the main factors for the diversified uses of Terminalia chebula as mentioned in traditional system of medicine. Theses findings give scientific basis for establishing the traditional uses of Terminalia chebula and a new direction further research work to establish traditional uses with respect to specific constituent of Terminalia chebula.
60 min. cycle was further subjected to three successive extractions of 90 min. each with methanol (50 ml). The final dilution of 1st cycle was made upto 10.0 ml while those of 2nd and 3rd cycles were made upto 5.0 ml. Aliquots (20 mL) of each dilution were subjected to H.P.L.C and contents of tannic acid, gallic acid, chebulinic acid and ethyl gallate were calculated in each case using the respective regression equations of the calibration plot and are reported in Table 2.
References
1. Bruhn, J.G. and Holmstedt, B. (1981). Natural products as medicinal
agents. Beal, J. and Reinhard, E., (Eds.), Hippokrates, Stuttgart, pp. 224235 2. 3. 4. 5. Cordell, G.A. (1995). Changing strategies in natural product chemistry. Phytochem., 40:1585-1612 Jossang, A., Seuleiman, M., Maidou, E. and Bodo, B. (1996). Pentacyclic triterpenes from Combretum nigricans. Phytochemistry, 41(2):591-594; Kundu, A.P. and Mahato, S.B. (1993). Triterpenoids and their glycosides from Terminalia chebula. Phytochemistry, 32 (4):999-1002 Lu, P.P. Liu, X., Li, X.G. and Wen, J.Q. (1991) Chemical constituents of fruits of Terminalia chebula Retz. Shanghai. Yike. Daxue. Xuebao., 18(3):233-235. Singh, C. (1990). 2-a-Hydroxymicromeric acid, a pentacyclic triterpene from Terminalia chebula. Phytochem., 29(7): 2348-2350.
Estimation of Tannic acid, gallic acid, ethyl gallate and chebulinic acid in Terminalia chebula samples
Accurately weighed powdered samples of 2.0 g of Terminalia chebula powder had been subjected to the optimized extraction procedure. Final dilutions of all the samples had been made upto 25.0 ml in methanol. Aliquots (20 mL) of the resulting solutions had been subjected to H.P.L.C. H.P.L.C. chromatogram has been represented in figure 2.

H.P.L.C method has been very effective in estimation of phytonutrients and there has been significant variation in
6.

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Simultaneous Estimation of Cetirizine Hydrochloride and Salbutamol Sulphate in Pharmaceuticals dosage forms by RP-HPLC
Dr. R.A. Singh*, Robin Kumar, Dinesh Kumar & A.K. Agrawal
Abestract: A simple, accurate, precise and reproducible method has been developed and validated for the Simultaneous estimation of Cetirizine Hydrochloride and Salbutamol Sulphate in Pharmaceuticals tablet dosage forms by RP-HPLC. The combination of both these drugs are available in the market. The literature survey revealed that there are number of methods have been reported for the estimation of Cetirizine and Salbutamol individually or in combination with other drugs however there is none of the method reported for the simultaneous estimation of these drugs in combined dosage forms. The separation was achieved using RPC-18 end capped (5mm) 250 x 4.0mm column in isocratic mode with mobile phase acetonitrile: water: dilute sulphuric acid (80: 16: 4 v/v) at a flow rate of 1.0ml/min, detection was carried out at 230nm using a UV detector. The retention time for Salbutamol was 1.7 mints and for Cetirizine was 2.67 mints. Dilutions of standard and sample were made in mobile phase. Recovery study was carried out by adding known quantity of standard drugs in the pre-analysed test solution and percentage recovery calculated in each case. The percentage recovery study for Cetirizine and Salbutamol was found to be 99.0 to 101.0% w/w respectively. Proposed method was found to be accurate, precise, simple, and rapid which could be easily used for routine analysis.
Introduction
Cetirizine Hydrochloride is a Piperazine derivative and metabolite of hydroxyzine, it is being used as non-sedative anti histamine drug. It is official in B.P.1 the colorimetric2, Spectrophotometric3, HPLC 4-5and HPTLC 6 Methods have been reported for the analysis of Cetirizine in pharmaceuticals preparation and biological fluids. Salbutamol sulphate is used for the treatment of Asthma and Chronic Bronchitis. Salbutamol is official in B.P.1 and I.P.7. Several HPLC8,9 Spectrophotometric 10,11 methods have been reported. The combination of both these drugs are available in the market as tablet dosage forms the literature survey revealed that there are number of methods have been reported for the estimation of Cetirizine and Salbutamol individually or in combination of other drugs however there is none of the method reported for the simultaneous estimation of these drugs in combined dosage forms. This paper present a simple accurate reproducible rapid and economic method for the simultaneous estimation of both drugs in combined dosage forms.
A Jasco Isocratic Liquid Chromatographic system, equipped with model PU-980 pump & Rheodyne injector with 20ml fixed loop and PU-975 UV-Detector. Chemicals & Reagents: Working Standard of Salbutamol Sulphate and Cetirizine Hydrochloride. Acetonitrile and Water of HPLC Grade and Sulphuric Acid of AR Grade. Mobile Phase: 800ml of Acetonitrile: 196ml of Water and 4ml of dilute Sulphuric Acid were mixed and filtered through 0.45ml fitter paper.
Standard Stock Solution:

a) Accurately weighed 60mg of Salbutamol Working standard was dissolved in 20ml of mobile phase in 50-ml volumetric flask and diluted to the mark (1 mg Salbutamol.ml-1). Accurately weighed 50mg of Cetirizine Hydrochloride working Standard was dissolved in 20ml of mobile phase in 50ml volumetric flask. Make the volume with mobile phase and filter through 0.45 m filter paper. (1mg.ml-1).
b)
*Author for correspondence: Director Technical - Arbro Pharmaceuticals, 4/9, Kirti Nagar Industrial Area, New Delhi. Email: arbrolab@arbropharma.com
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ANALYTICAL METHOD DEVELOPMENT Mixed Working Standard

Transfer 2ml of standard stock solution- (A) and 5ml of standard stock solution- (B) in a 50ml volumetric flask, mix and makeup volume with mobile phase mix and filter.
P TR
Table 1: Simultaneous Assay of Cetirizine Hydrochloride and Salbutamol in Dosage Formulation. Salbutamol Cetirizine Hydrochloride Claim Found Recovery Claim Found Recovery (mg) (mg)* (%)** (mg) (mg)* (%)** 1. 2.0 1.98 99.0 5.0 4.97 99.4 2. 2.0 1.97 98.5 5.0 4.95 99.0 *Average of three determinations. **After spiking the pre-analysed sample at different levels. and Cetirizine Hydrochloride in combined dosage form. The assay value & recovery data indicate that the method is free from interference of excipients and can be effectively used for routine quality control.
1.6 1.4
S. No.
Sample Preparation
20 Tablets were weighed and crushed to fine powder. A powder equivalent to 5mg of Cetirizine Hydrochloride was weighed accurately and dissolved in mobile phase and volume was made up to 50ml the solution was filtered through 0.45 m filter paper.
Chromatographic Conditions
Mode of separation : Reversed Phase- Isocratic Column : RPC-18 (5m) column 250 x 4.0mm prewashed with methanol (15 minutes) & equilibrated with mobile phase (30 minutes) prior to analysis. Mobile phase : 800ml of Acetonitrile: 196ml of Water and 4ml of dilute Sulphuric Acid were mixed and filtered through 0.45m fitter paper. Flow rate : 1.0ml/min Detector wave length : U.V. 230nm Chart speed : 1.0 Temperature : Ambient
Intensity (AU)
1.2
2.67, Cetirizine
1.0 0.8 0.6 0.4 0.2 0.0 0 1 2

1.70, Salbutamol
Procedure
The column was flushed with mobile phase till stable baseline obtained, 20ml of standard and sample dilution were injected separately under the chromatographic condition described above and scans were recorded each solution was run 5 times. The amount of Cetirizine and Salbutamol in sample was calculated by comparing the peek area ratio from standard.
10
Retention Time (Min.)

Typical HPLC chromatogram of sample containing Salbutamol sulphate and Cetirizine Hcl. For detailed chromatographic condition see text.
References
1. British Pharmacopoeia, Published by Her Majesty Stationary Office, 2003, London 2. Walily A.F.,korany M. A., Gindy A., and bedai M.F., J. of Pharma. & Biomed. Analy., 1998; 17(3), 435-442. 3. Parthasaradhin Reddy B., Suryanarayana M.V., venkatraman S., Satyanarayan Reddy M., and Sastry C.S., J. of Indian Drugs 1993; 30(6); 286-87. 4. Moncrieff J., J. of Chromato & Biomed. Appl.; 1992; 121(11); 128-130. 5. Rosseel M.T., and Lefebvre R.A., J. of Chrom. & Biomed Appl.; 1991; 565(4); 504-510. 6. Pandya k.k.Bhangru R.A, Gandhi T.P. et all, J.of Pharmacy and Pharmacol., 1996:48 (5):510-13. 7. Indian Pharmacopoeia, Volume I & II, Published by the Controller of Publication, New Delhi, 8. Shalaby A,J.liq. chromatogr. Relat. Technol. 1998,21(20), 3161-3171. 9. Joyce K.B, Jones A.E, Scott R.J, Biddlecombe R.A, 1998,12(23), 1899-1910. 10. Barky R.S, Rajak O.A, EIWalily A.F.M, Belal S.F, 1998, 14(3), 357-362. 11. Bakry R.S, Rajak O.A, EIWalily A.F.M, Belal S.F, 1995,28(14), 25032519.
Recovery
To establish the accuracy, reproducibility and precision of the proposed method, recovery experiments were carried out by spiking the pre-analysed sample at three different levels. The results are recorded on Table 1 below:
Results & Discussion

Under proposed chromatographic conditions, both the drugs, Salbutamol sulphate and Cetirizine Hydrochloride were well separated (Fig-1) with retention time 1.70 and 2.76 min respectively and the linearity was found in the range of 0.02mg.ml1 to 0.06mg.ml-1 for Salbutamol sulphate and 0.08mg.ml-1 to 1.2mg.ml-1 for Cetirizine Hydrochloride. The chromatogram was scanned at 230nm, being the absorption maxima of Salbutamol Sulphate the minor component in the formulations. A simple and accurate reversed phase HPLC method has seen proposed for simultaneous estimation of Salbutamol Sulphate

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ANALYTICAL METHOD DEVELOPMENT

ANALYTICAL METHOD DEVELOPMENT

Results and Discussion

ANALYTICAL METHOD DEVELOPMENT

Source: The Pharma Review, May 2009

ANALYTICAL METHOD DEVELOPMENT

Lifecare Innovations. *Author for correspondence E-mail: sales@lifecareinnovations.com

ANALYTICAL METHOD DEVELOPMENT

Results of analysis by both method were found almost same.

5.0 mg. 10.0 mg. 15.0 mg.

4.90 mg. 9.94 mg. 15.02mg.

98.0 99.4 100.1

2.50, 2.71 2.88, 2.98 3.45, 3.73

Procedure for Analysis of Powder for Injection

7.68 8.03 8.26

Typical Chromatogram of HPLC

Results and Discussion

Results of Analysis of commercial injection by proposed method

Source: The Pharma Review, March 2009

ANALYTICAL METHOD DEVELOPMENT

Fig. 1: Structural formula of Ceftazidime O S

ANALYTICAL METHOD DEVELOPMENT

3. Results and discussion

ANALYTICAL METHOD DEVELOPMENT

Fig. 2: Chromatogram of mixture of degradation solutions of Ceftazidime

C:\LALITHA.MET UV-VIS. 20 Dec. 2007 ch1: 21 Percent On Area

102.06 103.51 102.22 99.93

ANALYTICAL METHOD DEVELOPMENT 4. Conclusions

Source: The Pharma Review, March 2009

ANALYTICAL METHOD DEVELOPMENT

Materials and Methods

ANALYTICAL METHOD DEVELOPMENT

-------- (1) -------- (2)

Results and Discussion

300 Wave length (nm)

ANALYTICAL METHOD DEVELOPMENT

Method II % RSD % Recovery S.D.

Method III % % Recovery RSD S.D.

g/ % % Recovery ml Level S.D. OM 4 8 12 HTZ 2.5 5 12.5

150 100.150.404 0.404 100.630.195 0.194 99.690.950

average of three determinations

Source: The Pharma Review, December 2008

ANALYTICAL METHOD DEVELOPMENT

ANALYTICAL METHOD DEVELOPMENT

Result and Discussion

60.0 50.0 40.0 30.0 20.0 10.0 0.0

200.0 229 250.0

300.0 Wave length (nm)

Rf valve A - Amlodipine besilate B - Bisoprolol fumarate Fig. 2: Typical densitograms of AB and BF

ANALYTICAL METHOD DEVELOPMENT

Source: The Pharma Review, December 2008